-  £!£Cw 


BIOLOGY 

LIBRARY 

G 


AGEICULTUEAL 
BACTEEIOLOGT 


BY 

JOSEPH  E.  GREAVES,  M.S.,  PH.D. 

PROFESSOR  OF   AGRICULTURAL  BACTERIOLOGY  AND  PHYSIOLOGICAL   CHEMISTRY  IN  UTAH 

AGRICULTURAL  COLLEGE;     CHEMIST  AND  BACTERIOLOGIST  IN  UTAH 

EXPERIMENT  STATION. 


ILLUSTRATED  WITH  48  ENGRAVINGS 


LEA  &  FEBIGER 

PHILADELPHIA    AND    NEW    YORK 
1922 


Q  ft  5 

7 


COPYRIGHT 

LEA  &  FEBIGER 

1922 


PRINTED  IN  U.  S.  A. 


DEDICATED 
IN  LOVING  KEMEMBRANCE   OF 

PERNECY 

WHO  DURING  OTJR  FEW  SHORT  YEARS  TOGETHER 

CONSTANTLY  ENCOURAGED  AND 

INSPIRED  ME 


518885 


PREFACE. 


THE  organisms  considered  in  agricultural  bacteriology  are 
specifically  the  most  numerous,  chemically  the  most  active,  and 
economically  the  most  important  known.  This  being  true,  why  is 
so  much  interest  shown  in  the  injurious  and  so  little  in  the  beneficial 
bacteria?  There  are  two  chief  reasons  for  this  condition.  When 
an  outlaw  commits  some  crime  against  human  society  it  is  heralded 
far  and  near  and  the  machinery  of  the  law  is  set  in  operation  to 
apprehend  the  culprit  and  bring  him  to  justice.  So  it  is  with  these 
outlaws  in  bacterial  society.  The  typhoid,  or  perchance  some  other 
disease-producing  organism,  attacks  some  individual,  or  it  may  be 
an  entire  community.  If  it  be  typhoid,  we  hear  of  the  long-drawn- 
out  fight  between  the  human  individual  on  the  one  hand  and  the 
invisible  enemy  on  the  other.  If  disease  be  not  checked  it  spreads 
to  other  places,  and,  -as  in  the  Dark  Ages,  sweeps  like  a  prairie-fire 
over  a  whole  continent  or,  as  recently,  over  the  entire  world.  The 
second  reason  why  we  hear  more  of  the  disease-producing  organisms 
than  we  do  of  the  beneficial  bacteria  is  that  man  has  learned  that 
it  is  a  fight  between  him  and  .these  microbes  to  determine  which 
shall  inherit  the  earth .  He  has  learned  that  he  must  protect  him- 
self against  these  enemies.  For  these  reasons  man  has  studied  the 
bacterial  outlaw,  his  place  and  condition  of  growth. 

On  the  other  hand,  though  we  admire  the  magnificent  structures 
and  complex  institutions  which  have  been  reared  by  the  mind  and 
hand  of  men,  we  see  and  pass  on.  In  many  cases  we  do  not  stop  to 
contemplate  the  countless  millions,  living  and  dead,  who  have 
contributed  their  mite  that  things  might  be  as  they  are.  Man  does 
not  have  to  protect  himself  against  these  honest  toilers;  hence,  they 
go  unnoticed.  The  work  of  the  benefactor  lacks  the  sensationalism 
which  is  attached  to  that  of  the  destroyer.  So  it  is  with  the  count- 
less billions  of  beneficial  bacteria;  they  toil  on  day  and  night, 
generation  after  generation,  accomplishing  good  for  the  human  race. 
We  do  not  miss  them,  for  they  have  always  helped  us.  They  never 
become  discouraged,  but  work  for  our  good  until  conditions  become 
intolerable,  when  they  die  to  be  in  many  cases  replaced  by  the 
bacterial  outlaw. 

If  the  following  pages  help  to  systematize,  to  arouse  interest,  to 
stimulate  curiosity  or  inquiry  in  even  a  small  degree  in  this  intensely 


vi  PREFACE 

interesting  and  practical  subject,  the  author  will  feel  that  his  labors 
have  not  been  in  vain. 

It  is  coming  to  be  recognized  that  agricultural  bacteriology  and, 
agricultural  chemistry  are  at  many  points  intimately  associated. 
Hence,  the  writer  has  presupposed  a  knowledge  of  elementary 
chemistry  on  the  part  of  the  student.  However,  most  of  the  more 
complex  equations  have  been  grouped  in  one  chapter  so  they  may 
be  used  or  omitted  as  the  teacher  sees  fit. 

It  has  been  more  a  question  of  what  to  exclude  than  what  to 
include.  However,  the  writer  has  been  guided  throughout  by  the 
needs  of  the  student  of  agriculture,  and  hence  where  good,  complete 
volumes  are  available,  as  is  the  case  with  milk,  water,  sewage,  and 
some  other  subjects,  a  bare  outline  is  given;  so  the  student  should 
consult  other  works  for  a  more  exhaustive  treatment.  But  in  the 
case  of  soils  an  effort  has  been  made  to  go  more  into  detail.  Even 
in  these  chapters,  however,  no  attempt  has  been  made  to  review  all 
of  the  literature. 

In  the  preparation  of  this  work  I  have  drawn  freely  from  all 
available  sources.  Much  of  the  material  was  first  written  with  a 
complete  reference  to  the  literature,  but  it  soon  became  apparent 
that  such  a  procedure  would  produce  a  work  too  large  for  the  purpose 
for  which  this  was  written.  Hence,  all  references  have  been  elimi- 
nated. There  are,  however,  listed  at  the  end  of  most  chapters  a  few 
select  works  given  in  most  cases  because  of  the  references  which 
they  contain,  and  it  is  to  these  that  the  student  is  referred  for 
further  details.  At  the  end  of  the  last  chapter  is  given  a  list  of 
additional  works  which  have  been  consulted  in  the  preparation  of 
this  book. 

To  my  friends  and  colleagues  my  hearty  thanks  are  offered  for 
the  valuable  encouragement  and  assistance  given  in  the  preparation 
of  this  book.  I  am  under  particular  obligation  to  President  E.  G. 
Peterson,  Dr.  F.  S.  Harris,  Dr.  B.  L.  Richards,  Professors  George 
Stewart,  C.  T.  Hirst,  and  E.  G.  Carter  for  reading  parts  or  all 
of  the  manuscript  and  offering  many  helpful  suggestions,  also  to 
Mrs.  Blanche  C.  Pittman  for  her  painstaking  care  in  the  preparation 
of  the  manuscript  for  the  press. 

J.  E.  G. 

LOGAN,  UTAH,  1922. 


CONTENTS. 


CHAPTER  I. 

DEVELOPMENT  OF  BACTERIOLOGY 

Spontaneous  Generation 18 

Fermentation 21 

Smallpox .      .  24 

Anthrax 24 

Other  Work  of  Pasteur '.  26 

Other  Plagues  Conquered  ... .26 

Lister 26 

Yellow  Fever •.'. .'  .  '   .      .      .      .      .  26 

Agricultural  Bacteriology  .      .      .      .      .      .      . ,  27 

Future  Work .--'.. 27 

References    .  28 


CHAPTER  II. 

BACTERIA  AND  THEIR  PLACE  IN  NATURE 

Definition  of  Bacteria  .........;....  29 

Divisions  of  Plant  Kingdom    .... 30 

Occurrence  of  Bacteria       .      . •     .      .      .      .  31 

Role  of  Bacteria  in  Nature      .      .      .      .      .      .      ...      .      .      .  32 

Divisions  of  Bacteriology 36 


CHAPTER  III. 

MORPHOLOGY  OF  BACTERIA 

Bacilli V     ........  37 

Cocci       .  • 37 

Spirilla 38 

Gradations ...'.'.      .      .'     .      .      .  38 

Pleiomorphism 38 

Involution  Forms '.^ .      ...  38 

Size  and  Weight 39 

Brownian  Movement 40 

Organs  of  Locomotion 42 

Cell  Wall  Ectoplasm) 42 

Capsules 43 

Sheath 43 

Zoogloaa 43 

Cytoplasm 43 

Metachromatic  Granules 43 

Spores 44 

Longevity  of  Bacteria 45 


Vin  CONTENTS 


CHAPTER  IV. 

CLASSIFICATION  OF  BACTERIA 

Migula  Classification 46 

International  Rules  of  Botanicas  Nomenclature 49 

Classification  of  American  Bacteriological  Association       ....  50 

The  Class  Schizomycetes 50 

Order  Myxobacteriales 50 

Order  Thiobacteriales 50 

Order  Chlamydobacteriales 50 

Order  Actinomycetales 50 

Order  Eubacteriales  52 


CHAPTER  V. 

COMPOSITION  OF  BACTERIA 

Elementary  Composition 58 

Moisture 58 

Organic  Constituents 58 

Carbohydrates 58 

Extractives 58 

Proteins 59 

Inorganic  Constituents 61 

Variation  in  Composition  of  Different  Parts  of  Cell 61 

References 62 

CHAPTER  VI. 

FOOD  REQUIREMENTS 

Minimum  Requirements 63 

Maximum  Requirements 64 

Function  of  Food 64 

Source  of  Energy 64 

Moisture 65 

Osmotic  Pressure 67 

Kinds  of  Food  Required    .     . 67 

Carbon 68 

Nitrogen 68 

Hydrogen 68 

Sulphur 69 

Phosphorus .  69 

Potassium  I 69 

Other  Inorganic  Substances 69 

Oxygen  Requirements 69 

Vitamines 70 

References 70 

CHAPTER  VII. 

BACTERIAL  METABOLISM  ENZYMES 

Early  Theories  of  Fermentation 71 

Definition  of  Enzymes 72 

Terminology 75 

Properties  of  Enzymes .76 

Classification 79 

Hydrolytic  Enzymes 79 

Oxidizing  Enzymes 81 

References    .  81 


CONTENTS  ix 


CHAPTER  VIII. 

BACTERIAL  METABOLISM  PRODUCTS 

Physiologic  Classification 82 

Carbohydrate  Metabolism 82 

Acid  Production 84 

Acetic  Acid 85 

Lactic  Acid 85 

Butyric  Acid 86 

Other  Acid  Fermentations 87 

Oxidation  of  Organic  Acids 87 

Fats 87 

Products  from  Nitrogenous  Compounds 87 

Indol  and  Skatol 89 

Amins 90 

Pigments 91 

Chromophorous  Bacteria 93 

Chromoparous,  or  True  Pigment-forming  Bacteria  ...  93 

Parachrome  Bacteria 93 

Heat '  .  94 

Light , 94 

References 94 

CHAPTER  IX. 

INFLUENCE  OF  TEMPERATURE  AND  LIGHT  ON  BACTERIA 

Temperature  and  Speed  of  Reaction 95 

Relation  to  Heat 96 

Thermophilic  or  Heat-loving  Bacteria 96 

Psychrophilic  Bacteria 96 

Mesophilic  Bacteria 97 

Thermal  Death  Point 99 

Cold 100 

Light       . 101 

CHAPTER  X. 

EFFECT  OF  OTHER  AGENTS  ON  BACTERIA 

Radium  Rays "...  103 

Rontgen  Rays 103 

Electricity 103 

Drying 105 

Osmotic  Pressure 106 

Pressure 106 

Shaking 107 

CHAPTER  XI. 

EFFECT  OF  CHEMICALS  ON  BACTERIA 

Chemotaxis 108 

Disinfectants 110 

Laws  Governing  the  Actions  of  Disinfectants Ill 

Disinfectants  of  the  Chlorine  Group 114 

Formaldehyde 116 

Sulphur  Dioxide .            .  116 

Hydrocyanic  Acid  Gas .  117 

Mercuric  Chloride 117 

References .  117 


CONTENTS 


CHAPTER  XII. 

INFLUENCE  OF  ARSENIC  ON  BACTERIAL  ACTIVITY 

Occurrence  of  Arsenic 118 

Factors  Influencing  Solubility 118 

Ammonifiers 119 

Nitrification 119 

Nitrogen  Fixation 120 

How  Does  the  Arsenic  Act? 120 

References    .  126 


CHAPTER  XIII. 

EFFECT  OF  HEAT  AND  VOLATILE  ANTISEPTICS  ON  SOIL  BACTERIA 

Influence  on  Plant 127 

Effect  on  Properties  of  Soil 128 

Hypotheses  to  Account  for  Observed  Phenomena 133 

Koch's  "Direct  Stimulation"  Theory 133 

Hiltner  and  Stormer's  Indirect  Theory 133 

Russell  and  Hutchinson's  Protozoan  Theory 135 

Greig-Smith's  Bacteriotoxin  Theory 138 

References    .  ......  138 


CHAPTER  XIV. 

INFLUENCE  OF  SALTS  ON  THE  BACTERIAL  ACTIVITIES  OF  THE  SOIL 

Calcium  Carbonate 139 

Lime 141 

Gypsum 142 

Calcium  Chloride 142 

Iron  Sulphate     .      .      .      .    , . 

Magnesium  Salts 

Manganese 

Potassium  Salts 144 

Sodium  Salts 145 

Variation  in  Effect  Produced .146 

Stimulating  Action 147 

Toxicity  of  Various  Salts .149 

Reference                                           149 


HAPTER  XV.C 

INFLUENCE  OF  MANURE  ON  THE  BACTERIAL  ACTIVITIES  OF  THE  SOIL 

Number 151 

Ammonification  and  Nitrification 

Loss  of  Nitrates -  153 

Green  Manures 

Reference 159 

CHAPTER  XVI. 

THE  SOIL  FLORA 

Koch  Gelatin-Plate  Method 160 

Hiltner  and  Stonner  Dilution  Method    .      .      .      . 160 

Defects  of  Plate  Method 160 

Value  of  Bacterial  Counts 161 


CONTENTS  xi 

Number  of  Bacteria  in  Soil 161 

Factors  Influencing  Number ....  162 

Kinds  of  Microorganisms  in  Soil 164 

B.  megatherium  de  Bary 165 

Morphology 165 

Cultural  Characteristics 165 

Physiology 165 

B.  mycoides  Fliigge,  1886               .      .      .      . 166 

Morphology 166 

Cultural  Characteristics 167 

Physiology -.  167 

B.  cereus  Frankland,  1887        ...                                     ....  167 

Morphology 167 

Cultural  Characteristics 167 

Physiology        ...            168 

Ps.  fluorescens  (Flugge)  Migula 168 

Morphology 168 

Cultural  Characteristics 168 

Physiology 168 

Actinomyces •     .      .      .  169 

Reference 170 

CHAPTER  XVII. 

MINERALIZATION  AND  SOLVENT  ACTION  OF  BACTERIA 

Bacteria  as  Soil  Formers 171 

Calcium  Carbonate 172 

Phosphorus 173 

Sulphur 178 

Iron 180 

Potassium 180 

References 180 

CHAPTER  XVIII. 

THE  CARBON,  NITROGEN,  SULPHUR  AND  PHOSPHORUS  CYCLES 

The  Carbon  Cycle 182 

The  Nitrogen  Cycle 183 

The  Sulphur  Cycle "  .  184 

The  Phosphorus  Cycle 184 

References 187 

CHAPTERS  XIX. 

PUTREFACTION,  FERMENTATION  AND  DECAY 

Definitions 188 

Active  Agents 189 

Chemistry  of  the  Process .  192 

References 193 

CHAPTERS  XX. 
AMMONIFICATION 

Species  and  Distribution 196 

Methods 198 

Material  Ammonified 199 

Influence  of  Soil  and  Climatic  Conditions 200 

Moisture 200 

Aeration 202 

Lime  and  Magnesium 203 

Phosphorus 203 

Chemistry  of  the  Process 204 

References    .                                                                207 


xii  CONTENTS 

CHAPTER  XXI. 

NITRIFICATION 

Early  Theories 208 

The  Dawn  of  the  Biological  Theory .  209 

Isolation  of  Nitrifying  Ferments 211 

Distribution .                         .  217 

Reaction  of  Media 218 

Food  Requirements  of  Nitrifiers .  220 

Organic  Matter .  222 

Energy 223 

Metabolism 223 

Morphology .  225 

Influence  of  .Moisture 226 

Temperature 230 

Light  Rays 230 

Aeration  and  Cultivation 230 

Crop  and  Fallow 231 

Season 234 

Quantity  of  Nitrates  Formed 235 

Loss  of  Nitrates 236 

References 238 

CHAPTER  XXII. 

DENTRIFICATION 

Early  Theories 239 

Organisms  Concerned • 240 

Reaction  of  the  Media 241 

Food  Requirements 242 

Metabolism  of  Denitrifying  Organisms 243 

Influence  of  Water 244 

Temperature 245 

Losses  of  Nitrates  from  Manure  and  Soil 245 

Function  of  Denitrifiers 247 

References 247 

CHAPTER  XXIII. 

AZOFICATION 

Historical 248 

Distribution 252 

Reaction  of  the  Media 254 

Food  Requirements  of  the  Azofiers 256 

Organic  Soil  Constituents 259 

Influence  of  Colloids 260 

Sources  of  Energy  for  the  Azobacter 261 

Manure .      .  266 

Metabolism  of  Azotobacter 267 

Pigments  Produced  by  Azotobacter 270 

Morphology  of  the  Nitrogen-fixing  Organisms 271 

Methods .272 

Relation  of  Azotobacter  to  Other  Organisms 274 

The  Influence  of  Water 276 

Temperature ....  278 

Light  and  Other  Rays ' 281 

Aeration 281 

Season 281 

Crop 282 

Climate .      .  283 

Relationship  of  Azotobacter  to  Nitrate  Accumulations      ....  284 

Soil  Inoculation       . 284 

Soil  Gains  in  Nitrogen 287 

Reference 289 


CONTENTS  xiii 

CHAPTER  XXIV. 

SYMBIOTIC  NITROGEN  FIXATION 

Early  Theories 290 

Early  Observations  on  Root  Tubercles 291 

Species 292 

Cultural  Characteristics 297 

Morphology  of  the  Colonies 299 

Morphology  of  the  Bacteria 302 

Staining 303 

Bacteroids 303 

Mode  of  Entrance  into  the  Host        .            304 

Growth  of  the  Nodule .      .  304 

Relationship  to  Host 305 

Mechanism  of  Fixation  (Metabolism) 306 

Chemical 308 

Source  of  Energy 312 

Aeration        .      .      . 312 

Moisture 312 

Temperature 313 

Influence  of  Fertilizers 313 

Legumes  Associated  with  Non-legumes         314 

Soil  Gains  in  Nitrogen 315 

Soil  Inoculation 316 

Method  Involving  the  Use  of  One  Commercial  Culture     .      .      .      .317 

Alternative  Method 317 

Commercial  Cultures 318 

References 318 

CHAPTER  XXV. 

CROP  ROTATION 

Essential  Elements 319 

Element  Added  by  Legumes " 319 

Nitrogen        .''..' 320 

Rothamsted  Rotation •     .      .  321 

Nitrogen  Obtained  from  Atmosphere  by  Legumes        .      .      .      .      .  322 

Distribution  of  Nitrogen  in  Legumes .      .  323 

Legumes  Feed  on  Nitrates 323 

Nitrification  in  Soils 324 

How  to  Maintain  Soil  Nitrogen 325 

References    . .  326 

CHAPTERS  XXVI. 

CELLULOSE-DECOMPOSING  ORGANISMS 

Cellulose 327 

Early  Observations 327 

Work  of  Omelianski 330 

Morphology  and  Physiology    .            331 

Later  Work  on  Cellulose  Fermentation 333 

Function 333 

References ...  335 

CHAPTER  XXVII. 
BACTERIA  IN  AIR 

How  Bacteria  Enter  Air 336 

Number  and  Kind         336 

Factors  Governing  Number  and  Kind 337 

Bacteria  in  Inspired  and  Expired  Air -  339 

Air-borne  Infection                                                  339 


xiv  CONTENTS 


CHAPTER  XXVIII. 

WATER  BACTERIOLOGY 

Classification  of  Waters 340 

Numbers  of  Bacteria  in  Waters 342 

Sedimentation 343 

Light 343 

Temperature 344 

Food 344 

Classes  of  Bacteria 345 

Soil  Bacteria    .      . •    .  ,  .      .346 

Intestinal  Bacteria 347 

Natural  Purification  of  Water 347 

Artificial  Purification  of  Water .      .  348 

Chemical  Methods 349 

Ice  350 


CHAPTER  XXIX. 
WATER  AND  DISEASE 

Disease  Firs»t  Definitely  Proved  as  Due  to  Water 351 

Amount  of  Sickness  Due  to  Water 352 

The  Mills-Reincke  Phenomenon 353 

Cholera 354 

Typhoid 354 

References    .  359 


CHAPTER  XXX. 

SEWAGE  AND  SEWAGE  DISPOSAL 

Source,  Composition  and  Quantity  of  Sewage 360 

Bacteria  in  Sewage 361 

Hydrolyzing  Bacteria 362 

Oxidizing  Bacteria 363 

Reducing  Bacteria 363 

Pathogenic  Bacteria 364 

Necessity  of  Sewage  Disposal 366 

What  Should  Be  Accomplished  in  Sewage  Disposal? 366 

Methods  of  Dispoasal 366 

References 367 

CHAPTER  XXXI. 
MILK  BACTERIOLOGY 

Milk  as  a  Food 368 

Classes  of  Milk 371 

Bacteria  in  Milk 372 

Initial  Contamination 372 

Growth  of  Bacteria  in  Milk 374 

Changes  Produced  in  Milk  by  Bacteria '    .  375 

Abnormal  Changes  in  Milk 376 

Classes  of  Bacteria 376 

Acid-forming  Bacteria 376 

Peptonizing  Bacteria 377 

Bacteria  Producing  Milk  of  Unusual  Character 377 

Inert  Organisms 377 

Pathogenic  Bacteria 377 


CONTENTS  xv 


CHAPTER  XXXII. 

MILK  AND  DISEASE 

Sources  of  Infection 378 

Character  of  Milk-borne  Diseases      .      . .  379 

Extent  of  Milk-borne  Disease 380 

The  Tuberculin  Test 383 

Pasteurization 385 

References    . 386 

CHAPTER  XXXIII. 
BACTERIA  IN  OTHER  FOODS 

Bacteria  in  Butter 387 

Bacteria  in  Cheese 390 

Bacteria  in  Ice  Cream 390 

Bacteria  in  Condensed  Milk 391 

Bacteria  in  Bread 392 

Bacteria  in  Eggs 392 

Bacteria  in  Meat 393 

Bacteria  in  Canned  Foods 394 

References 394 

CHAPTER  XXXIV. 
BACTERIA  AND  FOOD-POISONING 

Classes  of  Food-poisoning 395 

Poisonous  Foods 395 

Metallic  Poisons ,      : 396 

Animals  Suffering  from  Disease    .      .      .      ...      ,    .  .  •    .      .      .      .  397 

Typical  Paratyphoid  Outbreaks   .      .      .      ......      .      .      .  397 

Offending  Foods ...      .      ...      ...      .  398 

Human  Infection .      .      .  399 

Ptomain  Poisoning 400 

Botulism 400 

Prevention 402 

References 403 

CHAPTER  XXXV. 

PRESERVATION  OF  FOOD 

Methods  of  Preserving  Food 404 

Cold 405 

Drying 406 

Pressure .406 

Canning 407 

Sugar  and  Salt 407 

Chemical  Preservatives 408 

References 410 

CHAPTER  XXXVI. 

BACTERIA  IN  THE  ARTS  AND  INDUSTRIES 

Alcoholic  Fermentation 411 

Vinegar 411 

Sauerkraut 412 

Ensilage 413 

Retting    ...'...• .414 

Tanning 414 

Vaccines 415 

References    .                                                                       .....  415 


AGRICULTUEAL  BACTERIOLOGY. 


CHAPTER  I. 
DEVELOPMENT  OF  BACTERIOLOGY. 

NOWHERE  in  the  whole  realm  of  human  endeavor  has  research 
been  crowned  with  more  glorious  achievements,  at  least  in  so  far 
as  the  welfare  of  the  human  race  is  concerned,  than  in  the  field  of 
bacteriology,  and  this  in  face  of  the  fact  that  bacteriological  research 
had  a  most  humble  and  recent  origin.  Even  the  dawn  of  bacteri- 
ology dates  back  only  to  the  last  quarter  of  the  seventeenth  century 
to  the  time  when  a  Dutch  linen-draper,  Anton  van  Leeuwenhoek, 
spent  his  leisure  time  in  grinding  lenses.  He  became  so  proficient 
in  this  that  his  lenses  were  superior  to  any  made  before.  Turning 
them  on  various  substances— raindrops,  saliva,  and  many  putrifying 
things— he  found  in  all  these  living,  moving  forms,  which  prior  to 
this  time  had  been  unrecognized.  We  can  imagine  his  joy  and 
surprise  from  this  statement:  "I  saw  with  wonder  that  my 
material  contained  many  tiny  animals  which  moved  about  in  a 
most  amusing  fashion.  The  largest  of  these  A  (Fig.  1)  showed 
the  liveliest  and  most  active  motion,  moving  through  the  water 
or  saliva  as  a  fish  of  prey  darts  through  the  sea;  they  were  found 
everywhere,  although  not  in  large  numbers.  A  second  kind  was 
similar  to  that  marked  B  (Fig.  1)  which  sometimes  spun  around 
in  a  circle  like  a  top.  These  were  present  in  larger  numbers  and 
sometimes  described  a  path  like  that  shown  in  C  to  D  (Fig.  1).  A 
third  kind  could  not  be  distinguished  so  clearly;  now  they  appeared 
oblong,  now  quite  round.  They  were  so  very  small  that  they  did  not 
seem  larger  than  the  bodies  marked  E,  and  besides  they  moved  so 
rapidly  that  they  were  continually  running  into  one  another.  They 
looked  like  a  swarm  of  gnats  or  flies  dancing  about  together.  I  had 
the  impression  that  I  was  looking  at  several  thousand  in  a  given 
part  of  the  water  or  saliva  mixed  with  a  particle  from  the  teeth  no 
larger  than  a  grain  of  sand,  even  when  only  one  part  of  the  material 
was  added  to  nine  parts  of  water  or  saliva.  Further,  the  greater 
part  of  the  material  consisted  of  an  extraordinary  number  of  rods, 
of  widely  different  lengths,  but  of  the  same  diameter;  some  were 
2 


18  DEVELOPMENT  OF  BACTERIOLOGY 

curved,  some  straight,  as  is  shown  in  F;  they  lay  irregularly  and 
were  interlaced.  Since  I  had  previously  seen  living  animalcules  of 
this  same  kind  in  water,  I  endeavored  to  observe  whether  there  was 
life  in  them,  but  in  none  did  I  see  the  smallest  movement  that  might 
be  taken  as  a  sign  of  life." 

This  patient  worker,  supplied  with  his  crude  microscope,  gives  a 
fairly  accurate  description  of  these  minute  forms  of  life.  But  this 
did  not  awaken  the  world  to  even  a  faint  realization  of  the  wonderful 
invisible  forms  of  life  which  were  present  in  everything  and  were 
always  working  for  good  or  evil.  It  did,  however,  revive  a  discussion 
which  had  waxed  long  and  furious  as  to  whether  life  can  spring 
spontaneously  from  inanimate  matter  or  whether  it  is  the  descendant 
of  preexisting  living  organisms. 


G 


FIG.  1. — The  first  drawings  of  bacteria  by  Leeuwenhoek.     The  dotted  line  C-D 
indicates  movement  of  the  organism.     (Morrey.) 

Spontaneous  Generation.— Back  in  the  sixteenth  century  a  famous 
physicist  and  chemist,  van  Helmont,  stated  that  mice  can  be 
spontaneously  generated  by  merely  placing  some  dirty  rags  in  a 
receptible  together  with  a  few  grains  of  wheat  or  a  piece  of  cheese. 
The  same  philosopher's  method  of  engendering  scorpions  is  also 
amusing. 

"Scoop  out  a  hole  in  a  brick,  put  into  it  some  sweet  basil.  Lay 
a  second  brick  upon  the  first,  so  that  the  hole  may  be  imperfectly 
covered.  Expose  the  two  bricks  to  the  sun,  and  at  the  end  of  a  few 
days  the  smell  of  the  sweet  basil,  acting  as  a  ferment,  will  change 
the  herb  into  a  real  scorpion." 

An  Italian,  Bouonami,  tells  of  a  wonderful  metamorphosis  which 
he  had  witnessed.  Rotten  timber,  rescued  from  the  sea,  produced 
worms;  these  gave  rise  to  butterflies;  and  strangest  of  all,  the  butter- 
flies became  birds. 

Everyone  thought  it  a  self-evident  fact  that  maggots  sprang 
spontaneously  from  decomposing  meat  or  cheese,  until  an  Italian 


SPONTANEOUS  GENERATION  19 

poet  and  physician,  Redi,  took  the  simple  precaution  of  screening 
the  mouth  of  jars  containing  meat  so  that  flies  could  not  enter. 
Flies  were  attracted  by  the  odor  and  deposited  their  eggs  on  the 
gauze,  and  it  was  from  these  that  the  so-called  "worms"  arose. 

The  theory  of  the  spontaneous  generation  of  mice,  scorpions,  and 
maggots  had  been  proved  untenable.  But  how  about  these  micro- 
scopic organisms  ?  They  surely  could  develop  directly  from  organic 
material.  For  now  anyone  provided  with  this  new  instrument,  the 
microscope,  could  easily  demonstrate  for  himself  the  spontaneous 
generation  of  microscopic  eels  in  vinegar,  or  produce  myriads  of 
different  and  interesting  living  creatures  in  simple  infusion  of  hay 
or  other  organic  material. 

Needham,  a  Catholic  priest,  evolved  the  theory  that  a  force  called 
"productive"  or  "vegetative"  existed  which  was  responsible  for 
the  formation  of  organized  beings.  The  great  naturalist,  Buffon, 
elaborated  the  theory  that  there  were  certain  unchangeable  parts 
common  to  all  living  creatures.  After  death  these  ultimate  con- 
stituents were  supposed  to  be  set  free  and  become  active,  until,  with 
one  another  and  still  other  particles,  they  gave  rise  to  swarms  of 
microscopic  creatures. 

Needham  in  1745  took  decaying  organic  matter  and  enclosed  it  in 
a  vessel;  this  he  placed  upon  hot  ashes  to  destroy  any  existing 
animalculse.  On  examining  the  contents  of  the  flasks  he  found  micro- 
organisms which  he  had  not  noted  at  first.  Later  (1769) ,  %>allanzani 
repeated  the  work.  He  felt  that  Needham  had  not  exercised  suffi- 
cient care  and  that  the  organisms  had  gotten  in  from  the  outside. 
Accordingly  he  boiled  the  material  for  one  hour  and  kept  it  in 
hermetically  sealed  flasks.  He  wrote:  "I  used  hermetically  sealed 
vessels.  I  kept  them  for  one  hour  in  boiling  water,  and  after  opening 
and  examining  their  contents  after  a  reasonable  interval,  I  found  not 
the  slightest  trace  of  animalculse,  though  I  had  examined  the  infusion 
from  nineteen  different  vessels." 

But  the  believers  in  the  theory  of  abiogenesis  were  not  convinced, 
for  they  claimed  that  the  boiling  altered  the  character  of  the  infusion 
so  that  it  was  unable  to  produce  life.  Voltaire,  with  his  characteristic 
satire,  took  up  the  fight  at  this  point  and  ridiculed  the  operations 
of  the  English  clergy  "  who  had  engendered  the  eels  in  the  gravy  of 
boiled  mutton,"  and  he  wittily  remarks:  "It  is  strange  that  men 
should  deny  a  creator  and  yet  attribute  to  themselves  the  power  of 
creating  eels."  But  this  was  a  controversy  to  be  settled  not  by 
ridicule  but  by  experimental  evidence. 

Spallanzani  answered  this  by  cracking  one  of  the  flasks  so  that 
air  could  enter.  Decay  soon  set  in.  Even  this  was  not  sufficient  to 
overthrow  a  popular  belief,  for  the  claim  was  made  that  the  sealing 
of  the  flasks  excluded  the  air,  and  air  was  essential  to  the  generation 
of  these  forms  of  life.  This  objection  was  answered  by  the  work  of 


20 


DEVELOPMENT  OF  BACTERIOLOGY 


\ 


many  ingenious  investigators.  Schulze,  in  1836,  passed  air  through 
strong  acids  and  then  into  boiled  infusions  and  failed  to  find  any 
living  organisms  in  the  infusion,  whereas  Schwann  passed  the  air 
through  highly  heated  tubes  with  the  same  results.  This  was  criti- 
cized by  their  opponents  who  claimed  that  the  chemical  alteration 


FIG.  2. — Experiment  of  Schulze:    Forcing  air  through  sulphuric  acid.     (Lafar.) 

of  the  air  subjected  to  such  drastic  treatment  had  been  responsible 
for  the  absence  of  bacteria  in  the  infusion.  The  work  of  Schroeder 
and  Dusch  (1853)  was  more  convincing,  for  they  found  that  it  was 
sufficient  to  stopper  the  bottles  with  cotton  plugs;  the  air  passed 
in  but  the  microorganisms  were  held  back  by  the  cotton  and  the 


FIG.  3. — Experiment  of  Schwann:     Heating  air  to  make  it  sterile.     (Lafar.) 

contents  of  the  flasks  kept  in  good  condition.  Every  now  and  then 
the  contents  of  a  flask  would  spoil,  even  after  it  had  been  carefully 
stoppered  and  boiled.  This  remained  a  stumbling  block  in  the  way 
of  those  who  maintained  that  life  sprang  only  from  life,  until  in  the 
year  1865  when  Pasteur  demonstrated  that  many  bacteria  may  pass 


FERMENTATION  21 

into  a  resting  stage,  and  while  in  this  condition  will  withstand  con- 
ditions which  quickly  kill  them  in  the  vegetative  stage.  Eleven 
years  later  Cohn  of  Breslau  carefully  investigated  organisms  in  this 
resting  or  spore  stage,  and  today  forms  of  microorganisms  are  known 
which  will  withstand  boiling  water  for  sixteen  hours  without  being 
killed,  and  others  resistant  enough  even  to  endure  for  many  hours  a 
10  per  cent,  solution  of  carbolic  acid. 

Fermentation.— Since  the  dawn  of  history  man  has  been  interested 
in  that  wonderful  process  known  as  fermentation,  and  although 
many  an  ingenious  theory  has  been  formulated  to  explain  it,  little 
more  than  theory  existed  unjfcilthe  classic  work  of  Pasteur  on  fer- 
mentation appeared  about  (f837y  Pasteur  claimed  that  all  forms  of 
fermentation  were  due  to  the  action  of  microscopic  organized  cells. 
An  idea  such  as  this,  even  at  this  late  date,  did  not  go  unchallenged, 
for  we  find  no  less  illustrious  workers  than  Helmholtz  and  Liebig 
opposing  it.  Liebig  scoffed  at  such  an  idea,  writing:  "Those  who 
pretend  to  explain  the  putrefaction  of  animal  substance  by  the 
presence  of  microorganisms  reason  very  much  like  a  child  who 
would  explain  the  rapidity  of  the  Rhine  by  attributing  it  to  the 
violent  motions  imparted  to  it  in  the  direction  of  Burgen  by  the 
numerous  wheels  of  the  mills  of  Venice." 

However,  Pasteur's  carefully  planned  experiments  soon  demon- 
strated that  without  the  microorganisms  there  would  be  no  fermen- 
tation, no  putrefaction,  no  decay  of  any  tissue,  except  by  the  slow 
process  of  oxidation.  The  care  with  which  his  experiments,  were 
planned  and  executed  are  well  shown  in  the  experiments  with  grape 
sugar,  concerning  which  he  wrote:  "I  prepared  forty  flasks  of  a 
capacity  of  from  two  hundred  and  fifty  to  three  hundred  cubic 
centimeters  and  filled  them  half  full  with  filtered  grape-must,  per- 
fectly clear,  and  which,  as  is  the  case  of  all  acidulated  liquids  that 
have  been  boiled  for  a  few  seconds,  remains  uncontaminated, 
although  the  curved  neck  of  the  flask  containing  them  remains 
constantly  open  during  several  months  or  years. 

"In  a  small  quantity  of  water,  I  washed  a  part  of  a  bunch  of 
grapes,  the  grapes  and  the  stalks  together,  and  the  stalks  separately. 
This  washing  was  easily  done  by  means  of  a  small  barber's  hair- 
brush. The  washing- water  collected  the  dust  upon  the  surface  of  the 
grapes  and  the  stalks  and  it  was  easily  shown  under  the  microscope 
that  this  water  held  in  suspension  a  multitude  of  minute  organisms 
closely  resembling  either  fungoid  spores  or  those  of  alcoholic  yeast, 
or  those  of  Mycoderma  vini,  etc.  This  being  done,  ten  of  the  forty 
flasks  were  preserved  for  reference;  in  ten  of  the  remainder,  through 
the  straight  tube  attached  to  each,  some  drops  of  the  washing- water 
were  introduced;  in  a  third  series  of  ten  flasks  a  few  drops  of  the 
same  liquid  were  placed  after  it  had  been  boiled;  and  finally  in  the 
ten  remaining  flasks  were  placed  some  drops  of  grape-juice  taken 


DEVELOPMENT  OF  BACTERIOLOGY 

from  the  inside  of  perfect  fruit.  In  order  to  carry  out  this  experi- 
ment the  straight  tube  of  each  flask  was  drawn  out  into  a  fine  and 
firm  point  in  the  lamp,  and  then  curved.  This  fine  and  closed  point 
was  filed  round  near  the  end  and  inserted  into  the  grape  while 
resting  upon  some  hard  substance.  When  the  point  was  felt  to 
touch  the  support  of  the  grape  it  was  by  a  slight  pressure  broken  off 
at  the  file  mark.  Then  if  care  had  been  taken  to  create  a  slight 
vacuum  in  the  flask,  a  drop  of  the  juice  of  the  grape  got  into  it;  the 
filed  point  was  withdrawn  and  the  aperture  immediately  closed  in 
the  alcohol  lamp.  This  decreased  pressure  of  the  atmosphere  in  the 
flask  was  obtained  by  the  following  means:  After  warming  the 
sides  of  the  flask,  either  in  the  hands  or  in  the  lamp  flame,  thus 
causing  a  small  quantity  of  air  to  be  driven  out  of  the  end  of  the 
curved  neck,  this  end  was  closed  in  the  lamp.  After  the  flask  was 
cooled,  there  was  a  tendency  to  suck  in  the  drop  of  grape-juice  in 
the  manner  just  described. 

"The  drop  of  grape-juice  which  enters  into  the  flask  by  this 
suction  ordinarily  remains  in  the  curved  part  of  the  tube,  so  that  to 
mix  it  with  the  must  it  was  necessary  to  incline  the  flask  so  as  to 
bring  the  must  into  contact  with  the  juice  and  then  replace  the  flask 
in  its  normal  position.  The  four  series  of  comparative  experiments 
produced  the  following  results: 

"  The  first  ten  flasks  containing  the  grape-must  boiled  in  pure  air 
did  not  show  the  production  of  any  organisms.  The  grape-must 
could  possibly  remain  in  them  for  an  indefinite  number  of  years. 
Those  in  the  second  series,  containing  the  water  in  which  the  grapes 
had  been  washed  separately  and  together,  showed  without  exception 
an  alcoholic  fermentation  which  in  several  cases  began  to  appear 
at  the  end  of  forty-eight  hours  when  the  experiment  took  place  at 
ordinary  summer  temperature.  At  the  same  time  that  the  yeast 
appeared,  in  the  form  of  white  traces,  which  little  by  little  united 
themselves  in  the  form  of  a  deposit  on  the  sides  of  all  the  flasks, 
there  were  seen  to  form  little  flakes  of  Mycelium,  often  as  a  single 
fungoid  growth  or  in  combination,  these  fungoid  growths  being 
quite  independent  of  the  must  or  of  any  alcoholic  yeast.  Often,  also, 
the  Mycoderma  vini  appeared  after  some  days  upon  the  surface  of 
the  liquid.  The  vibria  and  the  lactic  ferments,  properly  so-called, 
did  not  appear  on  account  of  the  nature  of  the  liquid. 

"  The  third  series  of  flasks,  the  washing- water  of  which  had  been 
previously  boiled,  remained  unchanged,  as  in  the  first  series.  Those 
of  the  fourth  series,  in  which  was  the  juice  of  the  interior  of  the 
grapes,  remained  equally  free  from  change,  although  I  was  not 
always  able,  on  account  of  the  delicacy  of  the  experiment  to  eliminate 
every  chance  of  error.  These  experiments  cannot  leave  the  least 
doubt  in  the  mind  as  to  the  following  facts: 

"  Grape-must,  after  heating,  never  ferments  on  contact  with  air, 


FERMENTATION 


23 


when  the  air  has  been  deprived  of  the  germs  which  it  ordinarily 
holds  in  a  state  of  suspension. 

"The  boiled  grape-must  ferments  when  there  is  introduced  into 
it  a  very  small  quantity  of  water  in  which  the  surface  of  the  grapes 
of  their  stalks  have  been  washed. 

"The  grape-must  does  not  ferment  when  there  is  added  to  it  a 
small  quantity  of  the  juice  of  the  inside  of  the  grape. 

"  The  yeast,  therefore,  which  causes  the  fermentation  of  the  grapes 
in  the  vintage-tub  comes  from  the  outside  and  not  from  the  inside 


FIG.  4. — Tyndall's  box.  One  side  is  removed  to  show  the  construction.  The 
bent  tubes  at  the  top  are  to  permit  a  free  circulation  of  air  into  the  interior.  The 
window  at  the  back  has  one  corresponding  in  the  front  (removed).  Through  these 
the  beam  of  light  sent  through  from  the  lamp  at  the  side  was  observed.  The  three 
tubes  received  the  infusion  and  were  then  boiled  in  an  oil  bath.  The  pipette  was 
for  filling  the  tubes.  (Popular  Science  Monthly,  April,  1877.) 

of  the  grapes.  Thus  it  destroyed  the  hypothesis  of  MM.  Trecol  and 
Fremy,  who  surmised  that  the  albuminous  matter  transformed 
itself  into  yeast  on  account  of  the  vital  germs  which  were  natural 
to  it.  With  greater  reason,  therefore,  there  is  no  longer  any  ques- 
tion of  the  theory  of  Liebig  of  the  transformation  of  albuminoid 
matter  into  ferments  on  account  of  the  oxidation." 

Pasteur's  work  did  not  stop  here,  for  he  soon  proved  that  a  disease 
that  was  attacking  the  silkworm  was  caused  by  bacteria.  And  from 
this  there  developed  the  idea  that  disease  in  general  is  due  to  bacteria. 


24  DEVELOPMENT  OF  BACTERIOLOGY 

If  there  were  any  doubts  left  in  the  minds  of  the  scientific  world 
as  to  the  fallacy  of  the  theory  of  spontaneous  generation,  after  the 
work  of  Pasteur,  they  were  dispelled  by  the  work  of  Tyndall. 
Tyndall  proved  that  in  an  atmosphere  devoid  of  dust,  as  on  the 
tops  of  mountains  and  in  some  ingeniously  constructed- boxes  used 
by  him,  perishable  substances,  such  as  beef  tea,  if  sterile  when  placed 
in  such  an  atmosphere,  will  keep  for  an  indefinite  period. 

Smallpox.— Smallpox  was  formerly  looked  upon  as  practically 
unavoidable  by  all  members  of  the  human  family,  as  is  seen  from  a 
popular  saying  current  in  Germany  in  the  eighteenth  century: 
"von  Pocken  und  Liebe  bleiben  nur  wenige  frei,"  from  smallpox  and 
love  few  remain  free. 

Concerning  smallpox  Macaulay  wrote  in  referring  to  the  death  of 
Queen  Mary  from  the  disease  in  1694:  "The  havoc  of  the  plague 
had  been  far  more  rapid;  but  plague  had  visited  our  shores  only 
once  or  twice  within  living  memory,  and  the  smallpox  was  always 
present,  filling  the  churchyards  with  corpses,  tormenting  with 
constant  fears  all  whom  it  had  not  yet  stricken,  leaving  in  those 
whose  lives  it  spared  the  hideous  traces  of  its  power,  turning  the 
babe  into  a  changeling  at  which  the  mother  shuddered,  and  making 
the  eyes  and  cheeks  of  the  betrothed  maiden  objects  of  horror  to  the 
lover." 

For  the  different  condition  which  exists  today  in  civilized  countries 
where  the  fear  of  smallpox  is  nearly  as  remote  as  that  of  leprosy, 
Edward  Jenner  (1749-1823)  is  chiefly  to  be  thanked.  His  attention 
was  at  first  directed  to  the  subject  by  the  remark  of  a  young  girl: 
"I  cannot  take  smallpox  for  I  have  had  cowpox."  After  consider- 
able labor  and  opposition  he  developed  arid  gave  to  the  world, 
without  monetary  consideration,  his  vaccine  which  has  all  but 
banished  from  the  wrorld  the  dreaded  disease— smallpox. 

Anthrax.— As  early  as  1863  investigators  had  seen  in  the  blood 
of  some  animals  that  had  died  of  a  disease  known  as  anthrax,  a  very 
small  rod-like  organism  which  permeated  all  the  capillaries.  Their 
experiments  showed  that  the  blood  from  such  an  animal,  when 
injected  into  the  veins  of  a  second  animal,  caused  it  to  die  of  the 
same  disease.  But  they  found  that  there  were  times  when  the  organ- 
ism could  not  be  discovered  in  the  blood  of  the  dead  animal,  although 
injection  with  blood  from  this  animal  would  cause  the  death  of 
another.  This  fact  left  a  doubt  in  the  minds  of  thinking  men  as  to 
whether  this  rod-shaped  organism  was  the  cause  of  the  animal's 
death  or  whether  it  was  "some  invisible  element  in  the  blood." 
Not  until  thirteen  years  later  was  this  fully  settled  by  the  work  of 
Robert  Koch.  He  not  only  saw  the  rod-shaped  organism,  but 
obtained  it  free  from  all  other  substances,  and  proved  that  it  was  the 
specific  cause  of  the  disease.  This  was  followed  by  many  other  dis- 
coveries, until  today  it  is  known  that  practically  all  diseases  are  due 


ANTHRAX  25 

to  microscopic  organisms.  Yes,  even  many  of  the  changes  taking 
place  in  the  body  and  associated  with  old  age  are  attributed  by  some 
writers  to  the  products  generated  by  bacteria. 

The  workers  in  this  field  are  not  satisfied  with  knowing  the  cause 
of  a  disease,  but  they  wish  to  know  how  they  may  ward  off  disease 
and  how  to  cure  it  when  once  it  gains  access  to  the  body  of  an  animal. 
Pasteur  soon  announced  that  he  had  found  a  preventive  for  anthrax. 
His  statement  was  immediately  challenged  by  the  president  of  an 
agricultural  society  in  such  a  way  that  it  was  brought  to  the  atten- 
tion of  the  entire  civilized  world.  He  suggested  that  the  subject  be 
submitted  to  a  decisive  public  test  and  offered  to  furnish  fifty  sheep, 
half  of  which  should  be  protected  by  the  attenuated  virus  prepared 
by  Pasteur.  Later  they  were  all  to  be  infected  by  the  disease- 
producing  organisms  and  if  the  vaccine  were  a  success  the  protected 
ones  were  to  remain  healthy,  the  unprotected  ones  to  die  of  the  dis- 
ease. Pasteur  accepted  the  challenge  and  suggested  that  for  two 
of  the  sheep  there  should  be  .substituted  two  goats,  and  that  there 
be  added  to  the  herd  ten  cows,  but  he  stated  that  these  latter  animals 
should  not  be  considered  as  falling  rigidly  within  the  test,  for  his 
experiments  had  not  yet  been  extended  to  cattle.  Before  this  time 
the  fame  of  Pasteur  had  been  considered  firmly  established,  but  now 
all  the  world  looked  on  with  doubt  to  think  that  any  man  should 
make  such  a  preposterous  claim.  On  May  5  the  animals  to  be  pro- 
tected received  their  first  treatment  with  the  vaccine  and  a  second 
two  weeks  later.  Virulent  cultures  of  the  disease-producing  organ- 
ism were  then  inoculated  into  the  animals.  The  results  of  the  test 
were  indeed  dramatic. 

"Two  days  later,  June  2,  at  the  appointed  hour  of  rendezvous,  a 
vast  crowd,  composed  of  veterinary  surgeons,  newspaper  corre- 
spondents, and  farmers  from  far  and  near,  gathered  to  witness  the 
closing  scenes  of  this  scientific  tourney.  What  they  saw  was  one  of 
the  most  dramatic  scenes  in  the  history  of  peaceful  science,  a  scene 
which  Pasteur  declared  afterward, '  amazed  the  assembly.'  Scattered 
about  the  enclosure,  dead,  dying,  or  manifestly  sick  unto  death,  lay 
the  unprotected  animals,  one  and  all,  while  each  and  every  protected 
animal  stalked  unconcernedly  about  with  every  appearance  of  per- 
fect health.  Twenty  of  the  sheep  and  the  one  goat  were  already 
dead;  two  other  sheep  expired  under  the  eyes  of  the  spectators;  the 
remaining  victims  lingered  but  a  few  hours  longer.  Thus,  in  a 
manner  theatrical  enough,  not  to  say  tragic,  was  proclaimed  the 
unequivocal  victory  of  science." 

It  has  been  estimated  by  conservative  writers  that  Pasteur's  dis- 
covery of  the  means  of  preventing  or  curing  anthrax,  silkworms' 
disease,  and  chicken  cholera,  adds  annually  to  the  wealth  of  France 
a  sum  equivalent  to  the  entire  indemnity  paid  by  France  to  Germany 
after  the  War  of  1870. 


26  DEVELOPMENT  OF  BACTERIOLOGY 

Other  Work  of  Pasteur.— This  was  only  a  part  of  the  work  of  this 
great  man,  for  in  1885  he  announced  a  cure  for  hydrophobia.  Prior 
to  this  time  the  disease  developed  in  at  least  16  per  cent,  of  the 
individuals  bitten  by  mad  dogs,  and  of  this  16  per  cent.,  100  per  cent, 
died.  Since  Pasteur's  discovery  the  number  of  deaths  from  this 
cause  has  been  reduced  almost  to  zero.  The  profound  importance 
of  his  work  has  been  well  summarized  by  Lord  Lister :  "  Truly  there 
does  not  exist  in  the  entire  world  any  individual  to  whom  the  medical 
science  owes  more  than  they  do  to  you.  Your  researches  on  fermen- 
tation have  thrown  a  powerful  beam  which  lightened  the  baleful 
darkness  of  surgery  and  has  transformed  the  treatment  of  wounds 
from  a  matter  of  uncertain  and  too  often  disastrous  empiricisms 
into  a  scientific  art  of  sure  beneficence.  Thanks  to  you,  surgery  has 
undergone  a  complete  revolution  which  has  deprived  it  of  its  terrors 
and  has  extended  almost  without  limit  its  efficacious  powers." 

And  Tyndall  writes:  "We  have  been  scourged  by  miserable 
throngs,  attacked  from  impenetrable  ambuscades,  and  it  is  only 
today  that  the  light  of  science  is  being  let  in  upon  the  murderous 
dominion  of  foes." 

Other  Plagues  Conquered.— In  the  realm  of  medicine  one  discovery 
after  another  has  followed  in  rapid  succession  during  the  last  few 
years,  until  today  diphtheria  instead  of  having  a  death-rate  of  over 
30  per  cent,  has  one  of  Jess  than  3.  Typhoid  fever  is  all  but  con- 
quered. Asiatic  cholera  and  the  yellow  fever  have  been  nearly 
wiped  from  the  face  of  the  earth,  thus  making  possible  the  building 
of  the  Panama  Canal. 

Lister.— Thanks  to  the  wonderful  work  of  Lord  Lister  we  no  longer 
have  that  terrible  suppuration,  which  before  his  time  followed  even 
slight  wounds.  At  the  close  of  the  nineteenth  century  it  was  asserted 
that  "Listerism"  had  saved  more  lives  than  had  been  sacrificed  by 
all  the  wars  of  the  nineteenth  century.  Although  continually 
brought  in  contact  with  suffering  and  misery,  this  truly  great  man 
did  not  lose  his  tender-hearted  nature  and  love  of  children,  as  is 
shown  by  the  following  story  related  by  one  of  Lister's  students. 

"One  day  when  Lister  was  visiting  his  wards  in  the  Glasgow 
Royal  Infirmary,  there  was  a  little  girl  whose  elbow-joint  had  been 
excised,  and  this  had  to  be  dressed  daily.  Lister  undertook  this 
dressing  himself.  The  little  creature  bore  the  pain  without  com- 
plaint, and  when  finished  she  suddenly  produced  from  under  the 
clothes  a  dilapidated  doll,  one  leg  of  which  had  burst,  allowing  the 
sawdust  to  escape.  She  handed  the  doll  to  Lister,  who  gravely 
examined  it;  then  asking  for  a  needle  and  thread,  he  sat  down  and 
stitched  the  rent,  and  then  returned  the  doll  to  its  gratified  owner." 

Yellow  Fever.— The  investigators  in  some  of  these  fields  have  gone 
into  it  not  only  with  a  knowledge  of  the  fact  that  failure  may  be 
their  lot,  but  they  even  risked  their  lives  in  the  work,  as  is  shown 


FUTURE  WORK  27 

in  the  fight  against  yellow  fever.  Dr.  Lazear,  an  American  army 
surgeon,  allowed  himself  to  be  bitten  by  a  mosquito  in  an  infected 
ward.  He  soon  acquired  yellow  fever  in  the  most  terrible  form  and 
died  a  martyr  to  science  and  a  true  hero.  He  gave  up  his  life  for 
others;  the  plain  record  of  his  sacrifice  is  recorded  thus  upon  a 
tablet  erected  to  his  memory:  "With  more  than  the  courage  and 
devotion  of  the  soldier,  he  risked  and  lost  his  life  to  show  how  a 
fearful  pestilence  is  communicated  and  how  its  ravages  may  be 
prevented."  That  this  is  conveyed  only  by  the  bite  of  the  mosquito 
was  shown  by  the  following:  Three  brave  men  slept  for  twenty 
nights  in  a  small,  ill- ventilated  room  screened  from  mosquitoes  but 
containing  furniture  and  clothing  smeared  with  the  excretion  of 
yellow  fever  patients — some  of  whom  had  died  of  the  disease.  None 
of  the  men  contracted  yellow  fever,  thus  indicating  the  disease  was 
not  of  a  contagious  nature. 

Agricultural  Bacteriology.— In  1883  Burrill,  by  the  discovery  of 
the  organism  which  causes  fire-  or  pear-blight,  opened  up  a  similar 
interesting  and  practical  field  in  "the  plant  kingdom  which  even  at 
the  present  day  is  only  in  its  infancy. 

It  may  appear  from  the  preceding  that  bacteria  are  all  enemies 
of  man,  but  this  is  not  true,  for  there  are  many  more  beneficial 
bacteria  than  injurious  ones. 

Even  in  the  field  of  agricultural  bacteriology  rapid  advances  have 
been  and  are  being  made.  To  Beijerinck,  Hellriegel,  Wilfarth,  Lip- 
man,  and  a  host  of  others,  we  owe  our  knowledge  concerning  the 
morphology  and  physiology  of  the  nitrogen-fixing  organisms.  In  1888 
Winogradsky  isolated  the  nitrifying  organisms  which  grow  on  a 
medium  devoid  of  all  organic  matter  and  since  that  time  there  is  an 
ever-increasing  volume  of  work  on  this  phase  of  the  subject.  Han- 
sen's  investigation  in  industrial  fermentation  is  also  important. 

Future  Work.— One  may  think  from  the  preceding  that  in  this 
field  of  science  there  is  little  to  be  done,  but  this  is  not  the  case,  for 
there  are  diseases  still  unconquered.  The  great  "  White  Plague" 
still  claims  its  millions  each  year.  There  are  diseases  which  are 
sapping  the  very  life-blood  of  the  nation,  yet  they  go  unchecked. 
Science  as  yet  has  not  come  to  the  aid  of  the  unfortunate  victims. 

As  regards  the  beneficial  organism  we  have  only  just  started  to 
realize  their  great  possibilities.  In  the  soil  are  five  great  classes  of 
organisms  which  deal  with  the  transformation  of  nitrogen.  One 
class  carries  on  putrefaction,  changing  the  insoluble  proteins  into 
ammonia,  another  picks  the  ammonia  up  as  formed,  transforming 
it  into  nitrites,  and  even  this  must  be  changed  into  nitrates  before 
plants  can  use  it.  Under  what  condition  are  these  changes  carried 
on  at  a  maximum  rate?  What  influence  has  moisture,  temperature 
crop,  and  method  of  tillage  on  this  change?  Some  of  these  questions 
are  being  answered  by  the  work  now  being  conducted,  but  there  are 


28  DEVELOPMENT  OF  BACTERIOLOGY 

many  yet  unanswered.  Still  they  are  vital  questions,  for,  in  many 
cases,  the  crop  yields  will  be  determined  by  the  skill  with  which 
these  various  changes  are  controlled.  There  is  another  set  of  organ- 
isms in  the  soil,  the  function  of  which  is  to  take  the  practically 
valueless  nitrogen  of  the  atmosphere  and  change  it  into  forms  such 
as  the  higher  plants  can  feed  upon.  How  may  we  control  them  for 
maximum  yields?  For  if  treated  properly  they  will  never  tire,  but 
toil  on  forever.  Then  again  it  is  possible  that  bacterial  action  may 
be  used  as  a  measure  of  soil  fertility  and  methods  so  perfected  which 
are  more  sensitive  than  any  now  in  use.  Truly,  in  this  field  great 
things  have  been  accomplished,  but  there  remains  yet  to  conquer 
fields  richer  by  far  than  the  workers  of  the  past  have  ever  dreamed. 

REFERENCES. 

Locy:     Biology  and  its  Makers. 

Paget:     Pasteur  and  after  Pasteur. 

Gregory:     Discovery — The  Spirit  and  Service  of  Science. 

Vollery-Radot:     The  Life  of  Pasteur. 

Libby:     History  of  Science. 


CHAPTER  II. 
BACTERIA  AND  THEIR  PLACE  IN  NATURE. 

BACTERIOLOGY  in  the  strictest  sense  is  that  branch  of  science 
which  deals  with  the  distribution,  morphology,  classification,  and 
function  of  bacteria.  However,  it  is  often  used  more  general  to 
include  bacteria,  yeasts,  molds,  and  protozoa.  A  better  term  where 
all  four  groups  are  included  is  "  microbiology/'  Many  of  the  modern 
writers  use  this  term. 

Definition  of  Bacteria.— Bacteria  are  extremely  minute,  simple, 
unicellular  organisms  which  multiply  with  great  rapidity,  usually 
by  transverse  fission,  and  are  devoid  of  chlorophyl.  Although  they 
contain  nuclear  material  which  is  usually  diffused  throughout  the 
cell  body  in  the  form  of  larger  or  smaller  granules,  they  possess  no 
definite  organized  nucleus.  They  are  generally  accepted  as  belong- 
ing to  the  vegetable  kingdom.  This  is  not  without  some  opposition, 
due  to  the  inherent  difficulty  of  the  subject,  as  is  so  admirably 
pointed  out  by  Fischer :  "  The  terms  '  animal'  and  '  plant'  are  collec- 
tive terms  invented  by  laymen  to  describe  familiar  living  things, 
insects  and  elephants,  mosses  and  oak  trees,  and  they  date  from  a 
time  when  such  minute  beings  as  bacteria  were  quite  unknown.  It  is 
therefore  as  superfluous  as  it  is  futile  to  attempt,  as  many  have  done, 
to  detect  the  distinguishing  characters  of  the  '  animal'  and  the 
'  vegetable'  kingdoms  among  organisms  for  which  these  terms  were 
never  intended.  For  this  reason,  Haeckel  and  others  have  proposed 
to  establish  a  third  dominion,  that  of  the  Protista,  which  shall 
include  all  those  forms  in  which  differentiation  has  not  been  pro- 
nounced on  the  lines  of  either  animal  or  plant  development.  The 
new  group  would  take  up  Radiolarians,  Flagellata,  and  Infusoria 
from  the  animal  side,  and  the  Cyanophycese  as  well  as  some  low 
forms  of  Algse  and  Fungi  from  the  plants.  The  border-line  between 
protista  on  the  one  hand  and  plants  and  animals  on  the  other  is— 
it  must  be  confessed— artificial.  To  these  protista,  which  embrace 
approximately  all  those  forms  of  life  we  commonly  call  micro- 
organisms or  microbes,  the  bacteria  belong." 

It  is  generally  stated  that  the  plant  cell  differs  from  the  animal 
cell  by  the  possession  of  a  firm  and  well  differentiated  wall,  wholly 
distinct  from  the  containing  protoplasm,  whereas  the  boundary 
surface  of  the  animal  cell  is  more  often  an  outer  layer  of  the  proto- 
plasm and  not  separable  from  it.  Moreover,  the  typical  cell  wall 


30 


BACTERIA  AND   THEIR  PLACE  IN  NATURE 


of  plants  is  usually  made  up  of  cellulose  or  one  of  its  derivatives;  the 
outer  membrane  of  the  animal  cell  is  nitrogenous  and  where  there 
is  a  heavy  cell  wall  it  is  chitinous.  Both  of  these  distinctions  break 
down  in  the  case  of  the  lower  forms  of  plant  and  animal  life. 

The  "blue-green"  algae,  or  Schizophycese,  possess  chlorophyll  and 
are  obviously  plants.  Structurally,  many  of  these  are  practically 
identical  with  bacteria.  This  constitutes  a  strong  argument  for  the 
plant  affinities  of  the  bacteria. 

Nor  is  it  an  easy  task  to  differentiate  nicely  between  bacteria, 
yeasts,  and  molds.  Generally  speaking,  typical  bacteria,  yeasts,  and 
molds  may  be  distinguished  from  each  other  as  follows:  Bacteria 
are  unicellular,  devoid  of  a  definite  organized  nucleus  but  con- 


tt!f 
ttffttfi 

iitsssft 


FIG.  5. — To  illustrate  the  close  relationship  of  the  bacteria  to  the  blue-green  algse. 
The  figures  to  the  left  (A)  are  blue-green  algae,  those  to  the  right  (B)  bacteria.  Those 
forms  most  closely  resembling  each  other  are  lettered  alike.  A,  blue-green  alga?: 
a,  Aphanocapsa;  b,  Merismopedia;  c,  Gleotheca;  d,  Spirulina;  e,  Phormidium;  /, 
Nostoc.  (All  adapted  from  West.)  B,  bacteria:  a,  Micrococcus;  b,  Sarcina;  c, 
Bacillus;  d,  Spirillum;  e,  Bacillus  in  chains;/,  Streptococcus.  (Buchanan's  Household 
Bacteriology.) 

taining  nuclear  material.  They  multiply  by  transverse  fission.  At 
times  they  are  united  into  filaments  or  masses,  but  are  usually 
easily  separated.  Yeast  cells  are  usually,  though  not  always,  larger 
than  bacteria.  Although  unicellular  they  contain  a  definite  organ- 
ized nucleus.  They  may  remain  united  after  cell  division,  but  each 
cell  constitutes  a  definite  entity.  Most  yeasts  multiply  by  budding 
—only  a  few  by  simple  fission.  Molds  are  multicellular,  nucleated 
organisms  which  are  usually  made  up  of  a  mass  of  interwoven  or 
radiating  threads  consisting  of  chains  of  cells. 

Divisions  of  Plant  Kingdom.— Plants  are  divided  into  four  great 
groups:  Spermatophytes  or  seed  plants,  Pteridophytes  or  fern  plants, 
Bryophytes  or  liver-worts  and  mosses,  and  Thallophytes  or  thallus 
plants.  This  last  group  ha.s  little  or  no  differentiation  of  vegetative 


OCCURRENCE  OF  BACTERIA  31 

organs,  such  as  stems  and  leaves.  Two  groups  stand  out  conspicu- 
ously—known as  algse  and  fungi,  but  there  are  other  groups  whose 
relationship  is  not  so  clear.  The  main  divisions  of  the  Thallophytes 
are  (1)  Myxomycetes,  commonly  known  as  slime  molds,  or  slime 
fungi,  which  combine  characters  of  plants  and  animals;  and  (2) 
Schizophytes  or  fission  plants,  characterized  by  cell  divisions  occur- 
ring in  rapid  succession  which  is  their  only  method  of  reproduction. 
They  consist  of  two  groups:  the  Cyanophycese,  or  blue-green  algae, 
and  the  Schizomycetes,  or  bacteria. 
The  relationship  is  shown  diagrammatically  below: 


Thallophytes — simple, 
undifferentiated  plants; 
do   not   develop   roots, 
stems  or  leaves. 


_f  Myxomycetes — slime  molds,  or  slime  fungi. 

(  Cyanophycese — blue-green 
Schizophytes — fission  plants  \      algse. 

[  Schizomycetes — bacteria. 
Algae,  including  seaweeds,  pond  scums,  water-silks,  etc.; 


contain  chlorophyll. 


f  Yeasts. 


I  Molds. 
Fungi  without  chlorophyll,    -j  Mildews. 

Smuts. 
[  Rusts,  etc. 

Occurrence  of  Bacteria.— Bacteria  are  ubiquitous,  occurring  as 
they  do  nearly  everywhere.  They  are  found  in  soil  to  great  depths, 
their  number  decreasing  with  the  depth  and  nature  of  the  soil,  being 
more  numerous  in  soil  containing  organic  matter  than  in  those 
practically  devoid  of  it.  Although  they  occur  in  the  atmosphere, 
it  is  not  their  normal  habitat,  for  growth  and  multiplication  cannot 
take  place  in  it  under  ordinary  conditions.  The  number  and  kind 
found  in  air  vary  with  a  number  of  factors,  chief  among  which  is 
locality.  The  air  of  some  high  mountains  is  practically  devoid  of 
bacteria;  city  and  country  air  also  differ  from  each  other  in  the 
number  and  kind  of  bacteria  they  contain.  Other  controlling 
factors  are  moisture,  presence  or  absence  of  injurious  substance,  and 
minute  particles  in  the  atmosphere. 

Most  natural  waters  contain  great  numbers  of  bacteria.  In 
sewage  and  polluted  water  they  are  especially  numerous,  but  occur 
only  in  small  numbers  or  not  at  all  in  deep  wells  and  springs.  The 
kind  of  organism  varies  with  the  composition  of  the  water  and  with 
the  original  contamination.  Milk  as  secreted  by  the  milk  glands 
of  cows  is  practically  free  from  bacteria,  but  the  vessels  in  which  it 
is  handled  so  contaminate  it  that  it  rapidly  gains  in  bacteria.  Often 
by  the  time  it  reaches  the  consumer  it  contains  millions  in  every 
cubic  centimeter.  In  short,  all  food  except  that  recently  cooked 
contains  bacteria,  the  number  and  kind  of  which  vary  with  the 
nature  and  age  of  the  food. 

Living  as  we  do  in  a  world  which  is  teeming  with  bacteria,  we  can 
expect  to  find  them  on  the  surfaces  of  the  skin  and  mucous  mem- 


32  BACTERIA  AND  THEIR  PLACE  IN  NATURE 

brane.  Normally,  the  infant  enters  the  world  free  from  bacteria, 
but  they  soon  begin  to  settle  on  the  skin;  they  penetrate  the  nose 
and  mouth;  the  first  respiratory  movements  and  cries  carry  them 
into  the  respiratory  passages;  and  between  the  tenth  and  seven- 
teenth hour  they  have  reached  the  intestines. 

Ordinarily,  the  deeper  respiratory  passages  contain  but  few 
bacteria,  but  it  has  been  proved  that  even  the  tubercle  bacillus  can 
penetrate  with  the  inspired  air  to  the  bottom  of  the  pulmonary 
alveoli. 

On  account  of  its  acidity,  yeasts  and  molds  nourish  better  in  the 
stomach  than  do  bacteria.  However,  at  least  thirty  species  of 
bacteria  (occurring  in  the  stomach)  have  been  described,  many  of 
which  have  attracted  special  attention  on  account  of  the  belief  that 
their  presence  may  favor  other  more  injurious  species. 

The  intestines,  on  account  of  their  alkaline  reaction  and  the  partly 
digested  condition  of  their  contents,  are  a  great  reservoir  of  bacterial 
activity.  Metchnikoff  and  others  have  given  an  immense  amount 
of  work  to  a  consideration  of  their  function  within  the  body  and  the 
probable  result  in  their  absence.  The  only  conclusion  which  is 
possible  at  present  is  that,  living  as  we  are  in  a  world  filled  with  micro- 
organisms, life  without  them  is  impossible.  All  that  can  be  done  is 
to  make  conditions  such  that  the  injurious  species  are  suppressed 
and  the  beneficial  ones  favored.  Out  of  this  has  grown  sour-milk 
therapy. 

The  normal  tissues  of  plants  and  the  blood  and  tissues  of  animals 
are  free  from  bacteria.  They  are  rarely  found  on  certain  healthy 
mucous  membranes,  such  as  those  of  the  kidney,  bladder,  and 
lungs.  Occasionally  they  pass  through  the  skin  or  .the  mucous 
membrane  of  the  digestive  tract  after  which  they  may  be  found  for 
a  short  time  in  the  blood.  This  is  especially  the  case  during  the 
height  of  digestion  and  it  probably  accounts  for  the  large  number 
of  leukocytes  which  swarm  in  the  intestinal  mucosa  and  which  have 
been  thought  to  be  in  some  way  associated  with  the  process  of  fat 
absorption. 

In  certain  diseased  conditions  the  blood  and  many  of  the  tissues 
of  the  human  body  are  found  to  contain  numerous  bacteria.  Soon 
after  death  even  the  saprophytes  rapidly  invade  and  decompose  the 
body  tissues. 

R61e  of  Bacteria  in  Nature.— Bacteria  play  a  wonderful  role  in  the 
many  transformations  going  on  in  this  world.  It  is  difficult  to  con- 
ceive of  life  without  them  and  their  help.  For  the  beneficial  ones 
we  turn  first  to  the  soil,  for  from  this— either  directly  or  indirectly— 
man  largely  draws  his  food,  clothing,  and  other  necessities  of  life. 
The  soil  is  not,  as  many  think,  a  dead,  inert  mass,  but  it  is  teeming 
with  life!  Both  microscopic  plants  and  animals  inhabit  it  by  the 
millions.  These  have  been  at  work  within  it  long  before  man  began 


ROLE  OF  BACTERIA  IN  NATURE  33 

to  till  the  soil.  In  the  formation  of  soil  from  the  primitive  rock, 
bacteria  played  no  small  part,  the  changes  wrought  by  the  elements 
first  giving  them  a  foothold. 

Changes  in  temperature  tear  loose  huge  rocks  and  break  them 
into  fragments.  It  is  well  known  that  most  substances  when  heated 
expand  and  contract  in  such  varying  degrees  that  parts  are  put 
under  a  strain.  This  strain  at  times  is  sufficient  to  cause  cracks  of 
various  sizes  to  occur  in  the  rock,  a  result  which  may  be  illustrated 
by  the  sudden  cooling  of  hot  glass  or  the  sudden  heating  of  cool 
glass.  Throughout  the  long,  hot  days  of  summer  rock  is  heated  to 
a  comparatively  high  temperature,  as  the  boy  who  has  chased  bare- 
foot over  their  surface  in  quest  of  grasshoppers  or  butterflies  will 
testify.  At  night  they  cool.  This  is  repeated  day  after  day.  This 
continued  heating  and  cooling  gradually  causes  small  crevices  to 
appear  in  even  the  most  resistant.  These  become  filled  with  water 
and  dust;  when  the  cold  nights  of  autumn  come  the  water  freezes. 
In  freezing,  the  water  expands  and  the  rocks  are  broken  into  pieces. 
So  it  continues  day  after  day  and  year  after  year,  until  the  rock 
becomes  .a  fine  powder.  Even  then,  however,  the  plant-food  is  still 
insoluble  and  cannot  be  taken  up  by  the  plant.  Long  before  it  has 
reached  the  form  of  powder,  bacteria  begin  to  grow  upon  the  surface 
of  the  rock  and  in  the  crevices.  In  their  growth  they  form  acids 
which  act  upon  the  insoluble  plant-foods,  rendering  them  soluble. 
Bacteria  continue  their  work  long  after  the  rocks  have  been  changed 
to  soil,  each  day  liberating  a  little  more  plant-food  for  the  growth 
of  plants  during  that  day.  During  the  year  the  bacteria  are  able  in 
a  fertile  soil  to  liberate  enough  plant-food  for  the  production  of  a 
good  crop.  When  manure  is  applied,  it  not  only  supplies  food  for 
the  growing  crop,  but  it  also  supplies  food  for  the  microorganisms, 
and  they  in  turn  liberate  more  of  the  insoluble  constituents  of  the 
fine  rock  particles  of  which  the  soil  is  mainly  composed.  There  are 
millions  of  them  in  every  ounce  of  soil,  struggling,  to  be  sure,  for 
their  very  existence",  but  always  rendering  a  little  more  mineral 
plant-food  available. 

One  of  the  essential  elements  for  crop  production,  and  the  one 
which  is  usually  in  the  soil  in  the  smallest  quantities,  is  nitrogen. 
This,  unless  it  be  applied  to  the  soil  in  the  form  of  the  costly  fer- 
tilizer, nitrates,  must  be  prepared  for  the  plant  by  bacteria.  The 
farmer  finds  his  crops  are  limited  directly  by  the  speed  with  which 
these  organisms  prepare  the  food  for  his  growing  crop.  If  they 
are  active,  other  things  being  favorable,  he  will  get  a  good  crop; 
but  if  they  do  not  play  their  part,  though  everything  else  may  be 
ideal,  yet  there  is  no  crop. 

Bacteriological  examinations  of  cultivated  soils  have  shown  that 
usually  those  that  are  richest  contain  the  greatest  number  of  bac- 
teria. The  number  in  the  soil  is  dependent  upon  the  quantity  and 
3 


34  BACTERIA  AND   THEIR  PLACE  IN  NATURE 

character  of  food  the  bacteria  find  in  the  soil.  If  the  soil  is  rich  in 
plant  residues— barnyard  manures  and  the  like— many  bacteria  will 
be  found  there  pulling  these  substances  to  pieces,  liberating  gases 
and  acids  which  act  upon  insoluble  particles  of  the  soil  and  render 
them  soluble.  One  class  of  organisms  changes  the  protein  constitu- 
ents of  the  soil  into  ammonia.  This  type  is  called  "ammonifiers." 
A  person  can  often  detect  their  activity  from  the  odor  of  ammonia 
coming  from  manure  heaps. 

Most  plants  cannot,  however,  use  nitrogen  in  the  form  of  ammonia; 
it  must  be  in  the  form  of  nitrates.  This  transformation  is  brought 
about  by  two  distinct  types  of  organisms.  One  of  them  feeds  upon 
the  ammonia  produced  and  manufactures  nitrous  acid.  Should  the 
change  cease  at  this  point  and  nitrites  accumulate  in  the  soil  in 
large  quantities,  plants  would  not  grow  upon  it,  for  this  is  a  poison 
to  plants.  But  in  soils  properly  cared  for  only  minute  quantities 
of  nitrites  accumulate.  As  soon  as  they  are  formed  another  type 
of  organism  feeds  upon  them  and  manufactures  nitric  acid  for  the 
growing  plant.  This,  when  formed,  reacts  with  other  constituents 
of  the  soil,  such  as  limestone.  It  is  then  ready  to  be  taken  up  by  the 
plant  and  manufactured  into  nourishing  food,  beautiful  flowers,  or 
fragrant  perfumes  for  the  human  family. 

Were  it  not  for  bacteria  the  world  in  time  would  be  filled  with 
never-changing  organic  matter.  The  plant  residues,  trees,  and 
animal  bodies  would  remain  stored  up  in  the  soil,  and  with  it  that 
element— carbon— which,  in  the  form  of  carbon  dioxid,  is  required 
by  all  chlorophyl  plants.  Bacteria,  in  getting  the  energy  which  they 
require  in  their  life  activity,  are  continually  liberating  carbon  so 
that  it  may  start  again  on  its  journey  of  construction.  If  carbon 
and  nitrogen  could  but  speak,  what  tales  of  wonderment  they  would 
tell !  The  chemist,  the  bacteriologist,  and  the  farmer  would  each  be 
wiser,  for  many  of  the  changes  through  which  carbon  and  nitrogen 
pass,  due  either  to  the  action  of  the  lower  plants— bacteria— or  that 
of  the  higher  plants  are  so  complex  that  even  the  scientist  with  his 
apparently  magical  methods  cannot  follow  them. 

So  far  only  the  plant-food  in  the  soil  and  the  changes  through 
which  it  passes  have  been  considered.  The  farmer,  however,  is 
usually  more  concerned  with  that  substance  his  soil  lacks  and  which 
must  be  supplied  in  order  to  get  good  crops.  In  many  cases  the 
lacking  element  is  nitrogen.  One  notes  from  the  fertilizer  quotations 
that  the  elements  will  cost  fifteen  cents  a  pound  or  over  if  purchased 
in  the  form  of  sodium  nitrate,  ammonium  sulfate,  or  dried  blood. 
If  one  stops  to  make  a  simple  calculation  he  finds  that  it  would  cost 
fifteen  dollars  for  enough  to  produce  100  bushels  of  corn,  eleven 
dollars  for  enough  to  produce  50  bushels  of  wheat,  and  seven  dollarsi 
and  fifty  cents  for  enough  to  produce  one  ton  of  alfalfa  hay.  In 
these  calculations  it  has  been  assumed  that  one  could  get  back  in 


ROLE  OF  BACTERIA  IN  NATURE  35 

the  form  of  corn,  wheat,  or  alfalfa  every  pound  of  commercial  nitro- 
gen that  haVheen  applied  to  the  soil,  which,  on  the  face  of  it,  is  an 
utter  impossibility.  So  we  have  to  look  to  other  means  of  getting 
nitrogen  for  our  growing  crop,  and  here  again  bacteria  come  to  our 
rescue.  ) 

There  are  seventy-five  million  pounds  of  atmospheric  nitrogen 
resting  upon  every  acre  of  land.  None  of  the  higher  plants,  however, 
have  the  power  of  taking  this  directly  out  of  the  air.  One  family 
of  plants,  the  Leguminosse,  in  which  are  included  peas,  beans,  alfalfa, 
clover,  and  many  others,  if  properly  infected  by  bacteria  have  the 
power  of  using  this  atmospheric  nitrogen.  Under  this  condition 
and  with  these  plants  nitrogen  no  longer  remains  the  limiting  ele- 
ment of  crop  production.  For  these  microscopic  organisms  which 
live  within  small  nodules  upon  the  alfalfa  are  master  chemists. 
Within  their  tiny  laboratory  they  can  bring  about  changes  which 
man  can  imitate  but  imperfectly  with  costly  machinery  and  under 
the  action  of  powerful  electric  currents.  In  some  of  the  experiments 
carried  on  at  the  Illinois  Experiment  Station  these  minute  organisms 
were  found  to  be  able  to  increase  the  value  of  the  first  cutting  of 
alfalfa  hay  $27.80  an  acre,  if  the  nitrogen  in  the  alfalfa  be  counted 
only  at  the  same  price  as  we  would  have  to  pay  on  the  market  for 
an  equivalent  -quantity  of  nitrogen  in  the  form  of  a  commercial 
fertilizer!  If  these  crops  be  plowed  under  the  fertility  of  the  soil 
would  be  increased  to  just  that  extent.  One  writer  has  said  of  them : 
"They  not  only  work  for  nothing  and  board  themselves,  but  they 
pay  for  the  privilege."  This  is  strictly  true,  for  all  they  require  is  a 
plant  on  which  to  grow  and  a  well-aerated  moist  soil  containing 
limestone.  They  cannot  work  in  an  acid  soil. 

There  is  another  class  of  nitrogen-gathering  organisms  within  the 
soil  which  differs  from  the  above  in  that  they  live  free  in  the  soil 
and  gather  nitrogen.  Under  ideal  conditions  they  may  gather 
appreciable  quantities. 

It  is  quite  possible  that  much  of  the  benefit  derived  from  the 
summer  fallowing  of  land  is  due  to  the  growth  within  the  soil  of  this 
class  of  organism  which  stores  up  nitrogen  for  future  generations 
of  plants.  It  has  been  found  that  they  are  more  active  and  found 
in  greater  numbers  in  such  a  soil.  All  the  work  that  the  farmer  puts 
upon  the  soil  to  render  it  more  porous  reacts  beneficially  upon  these 
organisms,  because  they  not  only  love  atmospheric  nitrogen  and 
oxygen,  but  must  have  them.  These  elements  are  absolutely  essen- 
tial to  their  life  activities  and  they  must  be  obtained  from  within 
the  soil  since  the  minute  organisms  cannot  live  upon  the  surface  for 
the  direct  rays  of  the  sun  kills  them  in  a  short  time. 

But  these  are  only  a  few  of  the  many  that  help  the  farmer.  They 
are  at  work  in  his  silo  rendering  the  feed  more  palatable  and  nutri- 
tious for  his  cattle.  They  are  working  in  his  milk  and  cream,  and 


36  BACTERIA  AND  THEIR  PLACE  IN  NATURE 

if  they  be  the  right  kind  they  give  to  butter  and  cheese  a  desirable 
flavor.  They  take  part  in  the  tanning  of  leather,  the  retting  of  flax, 
the  curing  of  tobacco,  and,  in  short,  they  help  us  in  a  hundred  and 
one  ways  we  little  suspect.  One  of  the  most  fascinating  and  instruc- 
tive tasks  set  for  man  is  to  learn  how  to  increase  the  work  of  the 
beneficial  bacteria  and  to  suppress  or  entirely  weed  out  the  injurious 
bacteria. 

Divisions  of  Bacteriology.— Bacteriology,  although  one  of  the 
youngest  of  sciences,  is  no  longer  confined  to  one  branch  which  can 
be  adequately  covered  by  one  text  or  its  whole  field  covered  by  any 
one  individual,  but  is,  as  are  the  other  sciences,  divided  into  a 
number  of  divisions  each  dealing  with  a  certain  phase  of  the  subject. 
The  main  divisions  are: 

1.  Agricultural  bacteriology  which  deals  with  the  bacteria  of  the 
soil  and  their  relation  to  plant  life. 

2.  Dairy  bacteriology  which  deals  with  the  bacteria  of  milk  and 
their  relations  to  dairy  products  such  as  pure  milk,  butter,  and 
cheese. 

3.  Industrial  bacteriology,  which  considers  the  use  of  bacteria  in 
the  arts  and  which  also  deals  with  methods  of  suppressing  injurious 
bacteria  and  favoring  the  beneficial. 

4.  Plant  pathology  which  deals  with  the  cause  and  prevention  of 
those  diseases  that  attack  plants  by  invading  their  tissues. 

5.  Animal  pathology  which  deals  with  bacteria  in  relation  to  the 
diseases  of  the  lower  animals. 

6.  Human  pathology  which  deals  with  the  distribution,  mor- 
phology, physiology,  and  pathological  changes  produced  by  bacteria 
which  are  pathogenic  to  man. 


CHAPTER  III. 
MORPHOLOGY  OF  BACTERIA. 

IN  shape,  bacteria  have  the  very  simplest  conceivable  structure, 
and  although  there  are  thousands  of  different  kinds  differing  in 
properties,  they  all  have  one  of  three  general  forms:  rod-shaped, 
spherical,  or  spiral. 

Bacilli.— The  rod-shaped  organisms,  which  may  be  compared  to 
a  lead  pencil,  are  cylindrical  organisms  in  which  a  longer  and  shorter 
dimension  may  be  recognized.  They  are  the  bacilli  (sing,  bacillus) . 
The  ends  of  the  organisms  may  be  convex,  less  often  flat  or  even 
concave.  The  size  also  varies,  some  being  so  short  that  it  is  next 
to  impossible  to  tell  whether  they  are  rods  or  globular  organisms; 
others  are  comparatively  long. 


03CQOOO 


FIG.  6. — The  normal  types  of  bacteria.  1-6,  cocci;  7-13,  bacilli;  14-16,  spirilla; 
1,  micrococcus;  2  and  8,  diplococci;  4>  tetracoccus;  5,  sarcina;  6,  streptococcus  (the 
lower  chain  includes  an  arthrospore) ;  7  and  8,  bacilli;  9,  10,  12  and  18,  bacilli  with 
various  granules;  11,  streptobacillus;  14,  vibrio;  15,  spirillum;  16,  Spirocheta  trepo- 
nema.  (Kendall.) 

Cocci.— The  cocci  (sing,  coccus)  are  typically  spherical  and  may 
be  likened  to  a  ball  or  at  times  to  an  egg.  They  may  in  the  early 
stages  of  cell  division  appear  temporarily  as  bacilli  with  convex  ends. 
They  often  occur  in  pairs,  diplococci,  in  which  case  usually  their 
proximate  surfaces  are  flattened.  This  flattening  of  the  organism 
may  at  times  be  accompanied  by  an  elongation  of  the  axis  of  the 
organisms  parallel  to  the  plane  of  opposition.  This  leads  to  the 
coffee-bean  shape  exemplified  in  the  gonococcus  and  miningococcus. 
At  other  times  we  have  the  flattening  perpendicular  to  the  plane  of 
the  flattened  surface  as  seen  in  the  "  lance-shaped"  pneumococcus. 
The  cocci  may  be  large  or  small  and  group  themselves  in  various 
ways. 


38  MORPHOLOGY  OF  BACTERIA 

Spirilla.— The  third  group  is  the  spirilla  (sing,  spirillum)  and  may 
be  likened  unto  a  corkscrew.  The  spiral  may  be  loosely  or  tightly 
coiled  or  there  may  be  one,  two,  or  many  coils.  At  times  the  curve 
may  be  so  slight  that  the  organism  viewed  under  the  microscope 
appears  "  comma-shaped. ' ' 

More  bacilli  are  known  than  cocci  and  more  cocci  than  spirilla. 
Migula  enumerates  833  bacilli,  343  cocci,  and  96  spirilla,  a  total  of 
1272.  Other  workers  have  tabulated  more  with  a  similar  propor- 
tional distribution  among  the  various  groups. 

Gradations.— The  difference  between  these  fundamental  types  is 
at  times  very  slight.  In  fact  the  cocci  often  merge  into  the  bacilli 
and  the  bacilli  into  the  spirilla.  It  is  often  difficult  accurately  to 
distinguish  between  the  various  groups,  as  is  exemplified  by  the 
fact  that  at  times  B.  prodigious  has  been  described  by  one  investi- 
gator as  a  coccus  and  at  another  time  by  a  different  worker  as  a 
bacillus.  This  same  condition  holds  for  the  pneumonia  germ  and  the 
one  causing  pear  blight,  whereas  the  cholera  organism  has  been 
described  both  as  a  bacillus  and  a  spirillum. 

Pleiomorphism.— By  pleiomorphism  is  meant  a  permanent  or 
semipermanent  change  in  the  normal  form  of  the  organism.  The 
organism  may  at  one  time  represent  a  coccus,  at  another  a  bacillus, 
and  at  still  another  a  spirillum.  This  led  the  early  writers  to  believe 
that  there  was  a  mutability  of  species.  The  condition  is  especially 
likely  to  occur  among  some  soil  organism  and  much  light  has  been 
thrown  on  the  subject  by  Lohnis  who  finds  the  life  history  of  bacteria 
to  be  only  slightly  less  complex  than  that  of  other  organisms. 


FIG.  7. — Involution  forms  from  bacilli.     (From  Fliigge.) 

Involution  Forms.— Although  the  form  of  bacteria  is  quite  constant 
under  normal  conditions,  yet  there  is  a  tendency  with  many  organ- 
isms, especially  when  grown  for  some  time  on  artificial  media,  to 
show  abnormal  or  bizarre  forms.  Such  organisms  are  known  as 
involution  forms.  Some  of  the  rod-shaped  organisms  may  appear 


SIZE  AND  WEIGHT  OF  BACTERIA  39 

as  clubs  many  times  larger  than  the  ordinary,  or  they  may  appear 
as  crosses  or  stars.  The  formation  of  large  club-shaped  organisms 
is  very  characteristic  of  the  organism  which  causes  diphtheria, 
whereas  the  formation  of  crosses,  stars,  and  the  like  is  characteris- 
tic of  the  organism  which  grows  in  the  roots  of  alfalfa.  Some  writers 
have  considered  them  degenerate  forms  and  compared  them  to  the 
"  lame  and  halt"  in  the  human  species.  This,  however,  is  hardly  an 
apt  illustration,  for  these  peculiar  shaped  organisms  have  all  of  the 
powers  possessed  by  others  and  if  they  found  their  way  into  the 
body  of  an  animal,  they  would  be  just  as  likely  to  produce  the  dis- 
ease which  is  characteristic  of  the  organism  as  would  the  ones  with 
the  normal  shape. 

In  some  cases  this  characteristic  has  served  as  a  valuable  aid  to 
the  differential  diagnosis  of  the  organism.  This  is  especially  true 
with  the  plague  bacillus  which,  when  grown  on  nutrient  agar  con- 
taining from  2.5  to  3.5  per  cent,  of  sodium  chlorid,  is  prone  to  give 
rise  to  involution  forms. 

Size  and  Weight.— The  unit  of  measurement  in  microscopy  is  the 
micron  (/A),  or  micromillimeter.  This  is  O.OQ1  of  a  millimeter  or 
approximately  2Tijo^  of  an  inch.  The  majority  of  the  organisms 
vary  from  0.2/z  up  to  30  jii  or  40ju.  They  are  smallest  in  the  case  of 
the  cocci  and  largest  in  the  case  of  the  spirilla. 

Although  there  is  a  great  variation  in  the  size  of  bacteria,  all  are 
extremely  small;  even  the  largest  are  not  visible  to  the  naked  eye. 
The  smallest  are  beyond  the  range  of  our  most  powerful  micro- 
scopes, and  others  appear  as  mere  dots.  The  Pfeiffer  bacillus,  the 
one  which  was  thought  to  cause  influenza,  are  rod-shaped  organisms, 
and  if  they  be  placed  end  to  end  it  would  take  fifty  thousand  of 
them  to  reach  one  inch,  or  it  would  require  about  fifteen  thousand  of 
the  bacteria  which  cause  typhoid  to  form  a  line  one  inch  in  length. 
Of  the  very  largest  known  it  would  require  seven  thousand  to  reach 
an  inch.  We  often  magnify  bacteria  one  thousand  times  and  then 
they  appear  as  dots  under  the  microscope,  but  if  we  would  magnify 
a  man  to  that  extent  he  would  appear  to  be  six  thousand  feet  tall 
and  fifteen  hundred  feet  wide.  Bacteria  are  so  small  that  at  times 
we  find  five  millions  in  a  small  drop  of  milk  and  yet  they  have 
plenty  of  room  to  move  about,  for  it  would  require  one  hundred  and 
twenty-five  billion  to  weigh  the  same  as  a  drop  of  milk. 

A  person  may  wonder,  since  bacteria  are  so  small,  how  they  can 
bring  about  such  enormous  changes,  for  it  takes  but  a  short  time 
for  them  to  tear  to  pieces  the  body  of  a  large  animal  that  has  died. 
All  know  how  fast  various  plants  and  fruits  decay  under  appro- 
priate conditions.  Decay  is  due  to  bacteria.  One  organism  could 
of  itself  bring  about  only  a  small  change,  but  they  multiply  with 
almost  inconceivable  rapidity.  The  bacilli  grow  until  they  have 
reached  a  certain  length,  then  divide  into  two,  and  these  in  turn 


40  MORPHOLOGY  OF  BACTERIA 

grow  to  maturity  and  then  divide.  Some  of  them  may  remain 
linked  together,  and  hence  appear  as  long  chains. 

In  the  case  of  the  cocci,  they  may  divide  into  two  and  remain 
linked  together  as  diplococci  or  a  great  many  may  remain  connected 
together,  thus  giving  the  appearance  of  a  string  of  beads;  Strepto- 
coccus. This  is  the  characteristic  of  the  common  blood-poison 
organism.  Other  spherical  shaped  organisms  divide  alternately  in 
two  planes  and  when  they  remain  connected  together  and  great 
masses  are  formed  they  resemble  a  bunch  of  grapes;  Staphylococcus. 
This  is  a  characteristic  of  the  common  boil-causing  organism.  Still 
others  of  the  spherical  organisms  divide  alternately  in  three  planes 
and  when  they  remain  connected  appear  very  similar  to  a  bale  of 
cotton;  Sar ciiia.  This  is  a  characteristic  of  many  of  the  organisms 
found  in  air. 

It  has  been  estimated  that  if  bacterial  multiplication  went  on 
unchecked,  the  descendants  of  one  cell  would  in  two  days  number 
281,500,000,000,  and  that  in  three  days  the  descendants  of  this 
single  cell  would  weigh  148,356,000  pounds.  It  has  been  further 
estimated  by  an  eminent  biologist  that  if  proper  conditions  could 
be  maintained  for  their  life  activity,  in  less  than  five  days  they 
would  make  a  mass  which  would  completely  fill  as  much  space  as 
is  occupied  by  all  of  the  oceans  on  the  earth's  surface,  if  the  water 
has  an  average  depth  of  one  mile! 

Even  in  the  face  of  these  assumptions  one  need  not  fear,  for  bac- 
teria have  been  on  this  earth,  and  have  been  multiplying  probably 
long  before  the  advent  of  man,  and  as  yet  the  earth  has  not  been 
filled  by  them.  This  is  due  to  there  being  a  struggle  among  them, 
just  as  there  is  among  higher  plants  and  animals.  One  knows  that 
if  wheat  be  sown  too  thick,  none  of  it  will  mature.  Sometimes  it  is 
a  lack  of  food,  other  times  a  lack  of  sunshine,  at  still  other  times  it 
is  a  lack  of  moisture  which  prevents  the  growth.  So  it  is  with 
bacteria,  the  food  or  water  may  give  out,  but  more  often  it  is  the 
products  which  they  form  that  prevent  them  from  continuing  to 
multiply. 

Brownian  Movements.— If  one  examines  under  a  microscope  a 
suspension  or  colloidal  solution  containing  particles  about  IAC  in 
diameter,  they  are  seen  to  be  in  motion  oscillating  through  a  dis- 
tance about  equal  to  their  own  diameter.  With  smaller  particles 
the  oscillation  is  much  greater  proportionately.  When  the  diameter 
is  about  4/i  the  motions  are  hardly  perceptible.  The  mean  velocity 
for  a  particle  of  platinum  weighing  2.5  x  10  ~15  gm.  has  been  estimated 
to  be  3  x  10  ~2  cm.  per  second  at  ordinary  temperature.  These 
smaller  particles  often  travel  in  straight  lines  and  suddenly  change 
their  direction.  Zsigmondy,  describing  the  movement  of  the  gold 
particles  in  a  gold  hydrosol,  compared  them  to  a  swarm  of  dancing 
gnats.  This  interesting  phenomenon  is  called  "  Brownian  Move- 


BROWNIAN  MOVEMENTS 


41 


merit"  from  Robert  Brown  (1773-1858),  an  English  botanist,  who 
first  observed  them  in  1827  when  studying  grains  of  pollen.  Observa- 
tions made  by  ingenious  methods  upon  the  Brownian  movements 


FIG.  8. — Spirillum  of  Asiatic  cholera, 
showing  single  flagellum.  (Kolle  and 
Zetnow.) 


FIG.  9. — Spirillum  volutans,  showing 
flagella  at  either  end  of  the  bacterium. 
(Herzog.) 


of  colloidal  suspensoids  are  exactly  what  the  kinetic  theory  indi- 
cates would  be  the  behavior  of  molecules  of  that  size.  Both  dead 
and  non-motile  bacilli  show  this  movement  as  do  also  small  particles 
freely  suspended  in  the  liquid.  However,  many  bacteria  show  a 


FIG.  10. — Bacillus  proteus  vulgaris,  showing  numerous  flagella  around  the  entire  body 
of  the  bacterium.     (Herzog.) 

true  independent  motion  and  if  watched  the  organism  will  be  found 
to  change  its  position  with  relation  to  other  organisms.  This  is 
known  as  "  vital  movement." 


42  MORPHOLOGY  OF  BACTERIA 

The  speed  with  which  they  travel,  being  magnified  to  the  same 
extent  as  are  the  organisms,  makes  them  appear  to  be  travelling 
with  enormous  speed,  insomuch  that  Leeuwenhoek,  who  first 
described  it,  stated  that  "they  seemed  to  tear  through  each  other." 
The  actual  speed,  however,  is  not  great  for  the  typhoid  bacillus 
may  travel  a  distance  of  4  mm.  or  about  2000  times  its  own  length 
in  one  hour,  whereas  the  cholera  spirillum  has  been  known  to  attain 
a  speed  of  18  cm.  per  hour.  Some  organisms  are  motile  if  grown  on 
one  cultural  media,  and  non-motile  if  grown  on  another ;  for  example, 
the  colon  bacillus  is  usually  motile  if  examined  from  young  cultures 
grown  on  gelatin  or  agar,  but  non-motile  if  taken  from  boullion. 

Organs  of  Locomotion.— The  protoplasmic  threads  called  organs 
of  locomotion  are  flagella  or  cilia.  A  cilium  differs  from  a  flagellum 
in  that  the  former  has  a  simple  curve  whereas  the  latter  has  a  com- 
pound curve,  like  a  whip  lash.  The  size,  the  number,  and  the 
arrangement  of  the  flagella  are  characteristic  of  the  organism. 
Most  bacteria  possess  flagella  rather  than  cilia.  Differences  exist  in 
respect  to  the  number  and  position  of  the  flagella  on  the  cell  body. 
Some  forms  possess  only  a  single  flagellum  at  one  pole  and  are 
called  monotricha,  others  a  flagellum  at  each  pole  (amj^jtricha), 
others  a  tuft  of  flagella  at  one  pole  (lophotricha),  otheiHtgella 
projecting  from  the  whole  body  of  the  cell  (peritricha) ;  a8^  s^i\\ 
others  possess  no  flagella  and  are  known  as  atricha. 


FIG.  11. — Pneumococci  with  unstained  capsules.  From  pneumonia  sputum, 
stained  with  carbol-fuchsin  and  differentiated  with  weak  acid  alcohol.  Magnifica- 
tion 1000.  (Karg  and  Schmorl.) 

Cell  Wall  (Ectoplasm).— The  cell  wall  is  the  slightly  differentiated 
outer  portion  of  the  cell  substance.  Many  writers  prefer  to  call  it 
"ectoplasm."  Early  in  the  history  of  bacteriology  it  was  con- 
sidered that  the  absence  of  cellulose  in  bacteria  indicated  that  they 
belonged  to  the  animal  rather  than  to  the  plant  kingdom.  But 
cellulose  or  hemicellulose  has  been  identified  in  bacteria  from  pus, 


METACHROMATIC  GRANULES  43 

7?.  subtilis,  tubercle  bacilli,  and  diphtheria  bacilli.  However,  the 
great  majority  of  the  organisms  contain  chitin,  a  substance  which 
on  hydrolysis  yields  glucosamin,  CH2OH(CHOH)3CHNH2CHO, 
and  acetic  acid.  Chitin  is  typically  animal  in  origin  and  for  this 
reason  some  have  argued  that  the  bacteria  belong  to  the  animal 
and  not  to  the  plant  kingdom.  The  flagella  probably  originate  from 
the  ectoplasm. 

Capsules.— Many  bacteria  possess  a  capsule  which  is  an  outgrowth 
of  the  cell  membrane  and  is  composed  of  mucin.  In  stained  cultures 
it  usually  appears  as  a  halo  surrounding  the  organism.  The  forma- 
tion of  a  capsule  is  not  confined  to  only  a  few  species,  some  writers 
arguing  that  under  appropriate  conditions  all  organisms  form  them; 
yet  the  so-called  capsulates  are  especially  prone  to  do  so.  Some 
organisms  produce  capsules  when  grown  on  one  media,  but  not  if 
grown  on  another.  Milk  especially  favors  the  formation  of  capsules. 

Sheath.— Often  a  distinct  tube  is  formed  in  which  is  inclosed  the 
chain  of  cells;  to  this  tube  is  given  the  name  "sheath."  It  is  espe- 
cially characteristic  of  some  of  the  trichobacteria  as  crenothrix  in 
which  there  is  a  deposition  of  iron.  Sometimes  these  become  fossil- 
ized, occurring  in  hugh  deposits  in  ferruginous  water. 

Zob'glcea.— Often  the  gelatinous  material  of  the  cell  causes  great 
masses  of  cells  to  adhere  to  each  other,  to  which  condition  is  given 
the  name  "zoogloea."  This  is  especially  characteristic  of  the  nitri- 
fying bacteria. 

Cytoplasm.— Chemical  analysis  of  the  cytoplasm  of  the  bacteria 
cell  shows  it  to  be  richer  in  nitrogen  and  phosphorus  than  are  the 
cells  of  higher  plants.  Moreover,  on  being  stained  the  cytoplasm 
appears  as  a  homogeneous  mass  filling  the  whole  cell,  thus  making 
it  certain  that  bacteria  do  not  possess  a  nucleus  in  the  ordinarily 
accepted  sense  of  the  term.  But  the  fact  that  the  organisms  stain 
so  readily  with  the  ordinary  nuclear  stains  has  led  some  to  believe 
that  the  organisms  are  made  up  mainly  of  nuclear  material.  This 
is  the  view  held  by  Zettnow  who  has  succeeded  in  staining  some  large 
spirilla  in  a  living  motile  condition.  Hence  the  idea  held  by  the 
majority  of  workers  at  the  present  time  is  that  the  bacterial  cell  is 
composed  of  small  quantities  of  cytoplasm  in  which  is  imbedded 
large  quantities  of  fragmented,  irregularly  distributed  chromatin. 

Metachromatic  Granules.— Some  bacteria  contain  various  granules 
within  the  cell  which  stain  differently  from  the  substance  of  the  cell 
body;  these  are  known  as  " metachromatic"  granules  or  " Babes- 
Ernst"  granules,  or  because  of  their  frequent  position  at  the  ends  of 
bacilli  as  polar  bodies.  Microchemical  examination  has  shown  them 
to  be  composed  of  various  substances:  fat,  sulphur  granules,  gly- 
cogen,  lecithin,  and  protein-like  compounds. 

Their  function  has  been  variously  interpreted.  Some  have  com- 
pared them  to  the  centrosomes  of  more  highly  specialized  cells. 


44  MORPHOLOGY  OF  BACTERIA 

Others  consider  there  to  be  a  relationship  between  the  richness  of 
the  cell  in  granules  and  its  virulence.  Hill,  however,  considers  that, 
inasmuch  as  nitrates  increase  the  nitrogen  assimilated  by  Azotobacter 
and  the  number  and  size  of  the  volutin  bodies,  they  bear  some 
relationship  to  the  organisms'  power  to  fix  nitrogen.  Although  it 


FIG.  12. — Successive  stages  in  division  of  Bacillus  diphtheria,  showing  relation  of 
line  of  division  to  metachromatic  granule.  Continuous  observation  of  living  bacillus 
drawn  without  camera  lucida.  (Williams.) 

is  quite  possible  that  they  may  possess  various  functions  in  differ- 
ent organisms,  the  majority  of  them  would  seem  to  be,  as  suggested 
by  Meyer,  reserve  food  materials  which  occur  in  the  cytoplasm  of 
the  cells  of  various  bacteria.  They  are  most  numerous  in  rapidly 
growing  young  cultures  and  usually  disappear  when  the  food  becomes 
scarce. 

m  -***,, 

FIG.  13. — Types  of  bacterial  spores.     (Kendall.) 

Spores.— Bacteria  possess  the  power  of  mobilizing  the  vital  parts 
of  their  body  into  a  much  smaller  space  than  they  occupy  during 
their  normal  life.  They  exclude  all  of  the  excess  moisture  and  sur- 
round themselves  by  a  tough  resistant  coat.  In  some  respects 
this  form  of  the  organism  resembles  the  seed  of  the  higher  plant 
and  we  speak  of  it  as  a  spore.  While  in  this  stage  they  will  with- 
stand many  conditions  which  would  quickly  prove  fatal  to  growing 
bacteria.  Some  of  them  can  withstand  the  temperature  of  boiling 
water  for  many  hours,  or  they  may  survive  treatment  with  strong 
carbolic  acid.  For  the  time  being  they  have  lost  the  power  of  mul- 
tiplying, but  they  are  still  alive  and  if  they  are  brought  into  appro- 
priate surroundings  they  will  change  back  into  normal  bacteria  just 
as  a  kernel  of  wheat  changes  into  the  young  plant  when  placed  in 
moist  soil.  It  is  indeed  fortunate  for  mankind  that  but  few  of  the 
disease-producing  organisms  form  spores.  There  are,  however, 
many  of  the  bacteria  which  cause  fruit,  meat,  and  various  other 
food  products  to  spoil,  which  do  form  very  resistant  spores  and  this 


LONGEVITY  OF  BACTERIA  45 

is  why  many  food  products  have  to  be  heated  for  such  a  long  time, 
or  to  such  a  high  temperature  to  keep  them. 

The  manner  of  formation  of  spores  within  the  body  of  the  organ- 
ism is  characteristic.  They  develop  within  the  cell  body  and  hence 
are  called  "  endospores."  They  are  formed  by  the  bacilli  and  spirilla, 
but  not  by  the  cocci.  The  beginning  of  spore  formation  is  marked 
by  a  granulation  of  the  cell  contents.  As  the  process  proceeds  the 
granules  become  larger  and  eventually  fuse  and  collect  at  one  por- 
tion of  the  cell  which  is  then  surrounded  by  a  spore  wall.  The  spore 
may  be  either  smaller  or  larger  than  the  mother  cell.  In  the  latter 
case  there  is  a  bulging  of  the  mother  cell.  The  spore  may  be  equa- 
torial, polar,  or  intermediate  within  the  cell  depending  on  its  position. 
When  situated  equatorially  and  larger  than  the  mother  cell  it  gives 
to  it  a  boat-shape  appearance  (clostridia) .  If  situated  at  the  pole 
and  large,  we  have  the  capitate  or  drumstick  appearance.  When 
bacteria  are  found  in  chains  and  spores  form  in  the  end,  there  is  a 
tendency  for  them  to  occur  in  adjacent  ends  of  contiguous  cells. 
A  cell  usually  forms  only  one  spore ;  hence,  this  cannot  be  considered 
a  process  of  reproduction. 

When  the  spores  are  brought  under  favorable  conditions  of  food 
supply,  temperature,  and  moisture  they  germinate.  The  process 
differs  according  to  species.  In  some  species  the  spore  ruptures  at 
the  pole  and  the  young  cell  emerges  in  such  a  way  that  its  long  axis 
is  in  the  same  direction  as  the  long  axis  of  the  spore,  thus  leaving 
the  spore  membrane  still  visible  at  one  of  the  poles.  In  other  species 
the  spore  germinates  equatorially  and  the  young  cell  emerges  with 
its  long  axis  at  right  angles  to  the  long  axis  of  the  spore.  In  still 
other  species  there  is  no  rupturing  of  the  spore,  but  germination 
occurs  by  a  gradual  elongation  and  absorption  of  the  spore. 

Longevity  of  Bacteria.— Due  to  their  method  of  multiplication 
there  is  no  such  condition  as  old  age  among  bacteria  since  both 
daughter  cells  are  similar  in  age  and  composition.  It  is  well  known 
that  while  in  the  spore  condition  many  organisms  can  survive  for 
over  two  decades.  Both  the  spore-forming  and  non-spore-forming 
organisms  have  been  obtained  from  soil  which  had  been  kept  in 
bottles  in  an  air  dry  condition  for  more  than  fifty  years.  Recently 
Sarcina  lutea  and  other  well-known  air  organisms  have  been  obtained 
from  a  Mastodon  uncovered  by  the  recession  of  the  ice  in  Siberia. 
This  animal  must  have  been  covered  for  hundreds  of  years.  This 
would,  therefore,  seem  to  indicate  that  the  longevity  of  bacteria 
may  be  extremely  great. 


CHAPTER  IV. 
CLASSIFICATION  OF  BACTERIA. 

THE  difficulties  inherent  in  the  classification  of  bacteria  are 
numerous  and,  due  to  the  small  simple  structure  of  the  organism, 
cannot  be  worked  out  on  a  purely  morphological  basis  as  is  the 
case  with  the  higher  plant.  Moreover,  physiological  characteristics, 
such  as  pigment  production  which  at  first  sight  may  appear  useful 
are  not  constant.  Even  morphology  of  bacteria  was  not  considered 
constant  until  1872  at  which  date  Cohn  established  upon  mor- 
phological bases  a  classification  which  with  minor  changes  has  been 
retained  until  the  present.  Bacteria  play  a  part  in  many  fields  of 
activity,  and  hence  the  criteria  whereby  they  are  recognized  vary 
greatly  according  to  the  art  or  science  in  which  they  are  studied. 
This  has  led  to  considerable  confusion  in  classification  and  nomen- 
clature as  is  so  admirably  pointed  out  by  Jordan. 

"The  present  nomenclature  of  bacteriology  may  be  criticized 
on  two  grounds:  first,  as  already  pointed  out,  for  the  unwieldy 
size  that  certain  'genera'  have  been  allowed  to  assume;  and  second, 
for  the  haphazard  way  in  which  trinomial  and  even  quadrinomial 
names  have  been  bestowed.  Such  names  can  be  properly  employed 
only  with  reference  to  subspecies  or  varieties;  and  designations,  like 
B.  coli  communis,  Granulobacillus  saccharobujxyicus  mobilis  non- 
liquefaciens  and  Micrococcus  acidi  paralactici  liquefaciens  Halensi, 
are  both  cumbersome  and  unscientific.  The  use  of  a  single  genus 
name  for  a  multitude  of  organisms  is  in  fact  responsible  for  the 
tendency  toward  trinomial  nomenclature,  and  the  remedy  for  both 
conditions  would  seem  to  lie  in  the  abandonment  of  such  a  term  as 
Bacillus  for  the  name  of  a  genus  and  the  frank  establishment  of 
new  genera  on  the  basis  of  physiological  characters,  such,  for  example 
as  distinguish  the  colon-typhoid  group  or  the  diphtheria  group  of 
bacilli.  Until  some  such  reform  in  -nomenclature  is  brought  about 
the  names  used  to  designate  different  kinds  of  bacteria  will  fail  to 
make  clear  the  group  relationships  which  undoubtedly  exist,  and 
will  continue  to  be  a  stumbling  block  to  all  students  of  the  subject." 

The  classification  most  commonly  accepted  at  the  present  day 
is  that  formulated  by  Migula.  This,  with  certain  modifications,  is 
given  below. 

Bacteria,  Schizomycetes,  fission  fungi  (chlorophyll-free),  cell  divi- 
sion in  one,  two  or  three  planes;  many  varieties  possess  the  power 
of  forming  endospores.  Whenever  motility  is  present,  it  is  due  to 
flagella,  or  more  rarely  to  undulating  membranes. 


CLASSIFICATION  OF  BACTERIA  47 

FAMILY  I.— Coccacece— cells  in  free  state  spherical;  division  in  one, 
two  or  three  planes;  endospore  formation  rare. 

Genus  I.— Streptococcus— cells  divide  in  one  plane  only  for  which 
reason;  if  they  remain  connected  after  fission  bead-like  chains  may 
be  formed;  no  organs  of  locomotion. 

Genus  II.—Micrococcus  (Staphylococcus)  — cells  divide  in  two 
planes,  whereby,  after  fission,  tetrad  and  grape-like  clusters  may  be 
formed;  no  organs  of  locomotion. 

Genus  III.— Sarcina—  cells  divide  in  three  planes,  whereby,  after 
fission,  bale-like  packets  are  formed;  no  organs  of  locomotion. 

Genus  IV.— Planococcus— cells  divide  in  two  planes,  as  in  Micro- 
coccus;  possess  flagella. 

Genus  V.—Planosarcina—  cells  divide  in  three  planes,  as  in  Sar- 
cina; posses  flagella. 

FAMILY  II.— Bacteriacece— cells  long  or  short;  cylindrical,  straight 
never  spiral;  division  in  one  plane  only,  after  preliminary  elongation 
of  the  rods. 

Genus  I. — Bacterium — cells  without  flagella ;  of  ten  with  endospores. 

Genus  II.— Bacillus— cells  with  peritrichal  flagella;  often  with 
endospores. 

Genus  III.— Pseudomonas— cells  with  polar  flagella;  endospores 
occur  in  a  few  species  but  are  rare. 

FAMILY  III.— Spirillacece— cells  spirally  curved  or  representing 
a  part  of  a  spiral  curve;  division  in  one  plane  only,  after  elongation 
of  cell. 

Genus  L— Spirosoma—  cells  without  organs  of  locomotion;  rigid. 

Genus  II.— Microspira— cells  rigid,  with  one  or  more  rarely,  two 
or  three  polar  undulated  flagella. 

Genus  III.— Spirillum— cells  rigid,  with  polar  tufts  of  five  to 
twenty  flagella  usually  curved  in  semicircular  or  flat  undulating 
curves. 

Genus  IV.— Spirochceta—  cells  sinously  flexible;  organs  of  loco- 
motion unknown,  perhaps  a  marginal  undulating  membrane. 

FAMILY  IV.— Chlamydobacteriacece— Forms  of  varying  stages  of 
evolution,  all  possessing  a  rigid  sheath,  which  surrounds  the  cells; 
cells  united  in  branched  or  unbranched  threads. 

Genus  I.  —  Streptothrix— cells  united  in  simple,  unbranched  threads; 
division  in  one  plane  only;  reproduction  by  non-motile  conidia. 

Genus  II.— Cladothrix— cells  united  or  pseudodichotomously 
branching  threads;  division  in  one  plane  only;  vegetative  multipli- 
cation by  separation  of  entire  branches;  reproduction  by  swarming 
forms  with  polar  flagella. 

Genus  III.— Crenothrix— cells  united  in  unbranched  threads; 
division  at  first  in  one  plane  only.  Later  the  cells  divide  in  all  three 
planes;  the  daughter  cells  become  rounded  and  develop  into  repro- 
ductive cells. 


48  CLASSIFICATION  OF  BACTERIA 

Genus  IV.— Phragmidiothrix— cells  at  first  united  in  unbranched 
threads,  dividing  in  three  planes,  thus  forming  a  rope  of  cells;  later 
some  of  the  cells  may  penetrate  through  sheath  and  thus  give  rise 
to  branches. 

FAMILY  V.—Beggisatoacea— cells  united  in  sheathless  threads; 
division  in  one  direction  of  space  only;  motility  by  undulating  mem- 
brane as  in  Oscillaria. 

Genus  L—  Thiothrix— unbranched,  non-motile  threads,  inclosed 
in  fine  sheaths;  division  of  cells  in  one  plane  only;  cells  contain 
sulphur  granules. 

Genus  II.— Beggiatoa—  cells  with  sulphur  granules. 

The  difficulties  inherent  in  this  classification  and  especially  the 
needs  of  reform  to  the*  agricultural  bacteriologist  are  seen  from  the 
following: 

"Many  workers  in  medical  bacteriology  and  in  other  special 
fields  of  applied  microbiology,  who  deal  with  only  a  few  well- 
recognized  species,  may  perhaps  feel  no  need  for  any  change  in 
current  practice.  Few  can  deny,  however,  that  it  is  a  serious 
inconvenience  for  such  names  as  B.  welchii,  B.  spprogenes,  B.  per- 
fringens  to  be  used  by  various  workers,  sometimes  for  the  same, 
sometimes  for  different  organisms,  or  for  the  same  form  to  be 
described  as  Bacterium  lactis  aerogenes  or  Streptococcus  lacticus 
when  it  is  isolated  from  milk  and  as  Streptococcus  salivarius  or 
Sir.  fecalis  when  it  is  isolated  from  the  human  mouth  or  intestine." 

"When  one  passes  from  a  study  of  the  practical  effects  of  the 
activity  of  some  particular  microbe  to  a  consideration  of  its  relation- 
ship to  other  forms  it  becomes  essential  not  only  to  have  a  name 
for  each  kind  of  organism  but  to  have  also  a  system  of  nomenclature 
which  will  make  it  possible  to  express  such  relationship  with  reason- 
able clearness  and  accuracy. 

"This  need  is  met  by  the  Linnaean  system  of  classification  uni- 
versally adopted  by  all  biologists  outside  our  own  limited  and  sys- 
tematically undeveloped  fields.  According  to  this  Linnaean  system 
each  recognizable  kind  of  plant  or  animal  receives  a  binomial 
Latinized  name,  the  first  half  designating  the  genus  or  group  to 
which  it  belongs  and  the  second  half  the  particular  kind  or  species 
to  which  the  name  applies.  The  genera  in  turn  are  grouped  in 
tribes,  the  tribes  in  families,  the  families  in  orders,  and  the  orders  in 
classes.  These  divisions  will  often  be  artificial  and  often  of  greatly 
unequal  size  and  importance  in  different  groups.  They  make  it 
possible,  however,  to  express  in  a  simple  manner  the  essential  facts 
of  biological  relationship— the  fact  that  A,  B,  and  C  are  more 
nearly  related  to  each  other  than  are  any  of  them  to  D,  E,  and  F; 
and  that  the  series  A-F  exhibits  common  relationships  closer  than 
any  similarities  which  its  members  bear  to  G  or  H. 

"If  such  a  system  is  accepted  it  is  in  the  next  place  important  to 


CLASSfFICATION  OF  BACTERIA  49 

make  sure  that  each  group,  from  species  to  class,  shall  bear  a  single 
universal  name.  The  name  need  not  be  appropriate;  it  need  only 
be  stable.  It  is  an  arbitrary  label,  not  a  description.  If  the  door 
be  once  opened  to  criticism  on  the  ground  of  inappropriateness, 
stability  must  disappear. 

"  It  is  in  order  to  ensure  uniformity  and  stability  of  nomenclature 
that  the  International  Codes  referred  to  have  been  formulated;  and 
it  is  to  the  International  Rules  of  Botanical  Nomenclature  (1910) 
that  we,  as  bacteriologists,  should  naturally  turn  for  guidance. 

"Leaving  out  a  great  many  minor  rules  and  recommendations, 
the  most  important  of  the  rules  which  would  affect  bacteriological 
practice  may  be  cited  as  follows: 

Chapter  I,  Article  7.— "Scientific  names  are  in  Latin  for  all 
groups." 

Chapter  II,  Article  10.— "Every  individual  plant  belongs  to  a 
species  (species),  every  species  to  a  genus  (genus),  every  genus  to 
a  family  (familia),  every  family  to  an  order  (ordo),  every  order  to  a 
class  (classis),  every  class  to  a  division  (divisio)." 

Chapter  III,  Section  1,  Article  15.— "Each  natural  group  of 
plants  can  bear  in  science  only  one  valid  designation,  namely,  the 
oldest,  provided  that  it  is  in  conformity  with  the  rules  of  Nomen- 
clature and  the  conditions  laid  down  in  Articles  19  and  20  of 
Section  2." 

Chapter  III,  Section  2,  Record  iii.— "Orders  are  designated 
preferably  by  the  name  of  one  of  the  principal  families,  with  the 
ending  ales." 

Chapter  III,  Section  3,  Article  21.— "Families  (families)  are 
designated  by  the  name  of  one  of  their  genera  or  ancient  generic 
names,  with  the  ending  acece" 

Chapter  III,  Section  3,  Article  23.  — "Names  of  subfamilies 
(subfamilies)  are  taken  from  the  name  of  one  of  the  genera  in  the 
group,  with  the  ending  oidecp.  The  same  holds  for  the  tribes 
(tribus)  with  the  ending  eos  and  for  the  subtribes  (subtribus)  with  the 
ending  ince." 

Chapter  III,  Section  3,  Article  24.— "Genera  receive  names 
(substantive  adjectives  used  as  substantives)  in  the  regular  singular 
number  and  written  with  a  capital  letter  which  may  be  compared 
with  our  own  family  names.  These  names  may  be  taken  from  any 
source  whatever  and  may  even  be  composed  in  an  absolutely 
arbitrary  manner." 

Chapter  III,  Section  3,  Article  26.— "All  species,  even  those  that 
singly  constitute  a  genus,  are  designated  by  the  name  of  the  genus  to 
which  they  belong,  followed  by  a  name  (or  epithet)  termed  specific, 
usually  of  the  nature  of  an  adjective  (forming  a  combination  of  two 
names,  a  binomial  or  binary  name)." 

Chapter  III,  Section  3,  Article  26,  Record  viii.— "The  specific 
4 


50  CLASSIFICATION  OF  BACTERIA 

name  should  in  general  give  some  indication  of  the  appearance,  the 
characters,  the  origin,  the  history  or  the  properties  of  the  species. 
If  taken  from  the  name  of  a  person  it  usually  recalls  the  name  of  the 
one  who  discovered  or  described  it,  or  was  in  some  way  concerned 
with  it. 

Chapter  III,  Section  3,  Record  x.— a  Specific  names  begin  with  a 
small  letter  except  those  which  are  taken  from  names  of  persons 
(substantives  or  adjectives)  or  those  which  are  taken  from  generic 
names  (substantives  or  adjectives)." 

The  classification  suggested  by  the  Committee  of  the  Society 
of  American  Bacteriologists  has  many  points  of  merit  to  the  agri- 
cultural bacteriologist.  The  classification  in  brief  is  as  follows : 

THE  CLASS  SCHIZOMYCETES. 

Minute,  one-celled  chlorophyll-free,  colorless,  rarely  violet-red  or 
green-colored  plants,  which  typically  multiply  by  dividing  in  one, 
two,  or  three  directions  of  space.  The  cells  thus  formed  are  usually 
spherical,  cylindrical,  comma-shaped,  spiral,  or  filamentous  and  are 
often  united  into  filamentous,  flat,  or  cubical  aggregates.  Filament- 
ous species  often  surrounded  by  a  common  sheath.  The  cell  plasma 
generally  homogeneous  without  a  morphologically  differentiated 
nucleus.  Reproduction  by  simple  fission.  In  many  species  resting 
bodies  are  produced,  either  endospores  or  gonidia.  Cells  may  be 
motile  by  means  of  flagella. 

A.  Order  Myxobacteriales.— Cells  united  during  the  vegetative 
stage  into  a  pseudoplasmodium  which  passes  over  into  a  highly- 
developed  cyst-producing  resting  stage. 

B.  Order  Thiobacteriales.— Cells  free  or  united  in  elongated  fila- 
ments.   Typically  water  forms,  not  cultivable  on  ordinary  media. 
Life  energy  derived  mainly  from  oxidative  processes.     Cells  typi- 
cally containing  either  granules  of  free  sulfur  or  bacteriopurpurin 
or  both,  usually  growing  best  in  the  presence  of  hydrogen  sulphid. 

C.  Order  Chlamydobacteriales. — Cells  normally  united  in  elongated 
filaments,  often  snowing  false  but  never  true  branching.    Typically 
water  forms.    Sulphur  and  bacteriopurpurin  are  absent.    Iron  often 
present  and  usually  a  well-marked  sheath. 

D.  Order  Actinomycetales. — Cells  usually  elongated^  frequently 
filamentous  and  with  a  decided  tendency  to  the  development  of 
branches,  in  some  genera  giving  rise  to  the  formation  of  a  definite 
branched  mycelium.    Cells  frequently  show  swellings,  clubbed,  or 
irregular  shapes.    No  pseudoplasmodium.    No  deposits  of  free  sul- 
phur or  iron.   No  bacteriopurpurin.   Endospores  not  produced,  but 
conidia  developed  in  some  genera.    Usually  (Tram-positive.    Non- 
motile.     Some  species  are  parasitic  in  animals  or  plants.     Not 
water  forms.     Complex  proteins  frequently  required.     As  a  rule 


CLASSIFICATION  OF  BACTERIA  51 

strongly  aerobic  (except  for  some  species  of  Actinomyces  and  the 
genera  Fusiformis  and  Leptotrichia)  and  oxidative.  Growth  on 
culture  media  often  slow;  some  genera  show  mold-like  colonies. 

FAMILY  l.—Actinomycetacece.— Filamentous  forms  often  branched 
and  sometimes  forming  mycelia.  Conidia  sometimes  present. 
Some  species  parasitic. 

Genus  1 . — A  ctinobacillus. — Filament  formation,  resembling  strep- 
tobacilli.  In  lesions  no  mycelium  formed,  but  at  peripheries  finger- 
shaped  branched  cells  are  visible.  Gram-negative.  Not  acid-fast. 
Type  species,  Act.  Lignieresi. 

Genus  2.— Leptotrichia.— Thick,  long,  straight  or  curved  threads, 
unbranched,  frequently  clubbed  at  one  end  and  tapering  to  the  other. 
Gram-positive  when  young.  Threads  fragment  into  short,  thick 
rods.  Anaerobic  or  facultative.  Non-motile.  Filaments  sometimes 
granular.  No  aerial  hyphse  or  conidia.  Parasites  or  facultative 
parasites.  Type  species,  Lep.  buccalis. 

Genus  3.—  A ctinomyces.— Organism  growing  in  form  of  a  much- 
branched  mycelium  which  may  break  up  into  segments  that  func- 
tion as  conidia.  Sometimes  parasitic,  with  clubbed  ends  of  radiating 
threads  conspicuous  in  lesions  in  animal  body.  Some  species  'are 
micro-aerophilic  or  anaerobic.  Non-motile.  Type  species,  Act. 
bovis  Harz. 

Genus  4. — Erysipelothrix. — Rod-shaped  organisms  with  a  ten- 
dency to  the  formation  of  long  filaments  which  may  show  branching. 
The  filaments  may  also  thicken  and  show  characteristic  granules. 
No  spores.  Non-motile.  Gram-positive.  Do  not  produce  acid. 
Micro-aerophilic.  Usually  parasitic.  Type  species,  Bacillus 
rhusiopathice  suis  Kitt  1893;  Mycobacterium  rhusiopathice  Chester 
1901;  Erysipelothrix  porci  Rosenbach  1909,  the  causal  organism  of 
swine  erysipelas. 

FAMILY  II.— Mycobacteriacece.— Parasitic  forms.  Rod-shaped, 
frequently  irregular  in  form  but  rarely  filamentous  and  with  only 
slight  and  occasional  branching.  Often  stain  unevenly  (showing 
variations  in  staining  reaction  within  the  cell).  No  conidia. 

Genus  I.— Mycobacterium.— Slender  rods  which  are  stained  with 
difficulty,  but  when  once  stained  are  acid-fast.  Cells  sometimes 
show  swollen,  clavate,  or  cuneate  forms,  and  occasionally  even- 
branched  cells.  Non-motile.  Gram-positive.  No  endospores. 
Growth  on  media  slow.  Aerobic.  Several  species  pathogenic  to 
animals.  Type  species,  Mycobacterium  tuberculosis. 

Genus  2.— Corynebacterium.— Slender,  often  slightly  curved,  rods 
with  tendency  to  club  and  pointed  forms,  branching  cells  reported 
in  old  cultures.  Barred  uneven  staining.  Not  acid-fast.  Gram- 
positive.  Non-motile.  Aerobic.  No  endospores.  Some  pathogenic 
species  produce  a  powerful  exotoxin.  Characteristic  snapping 
motion  is  exhibited  when  cells  divide.  Type  species,  Cornynebac- 
terium  diphtherioe. 


52  CLASSIFICATION  OF  BACTERIA 

Genus  3.— Fusiformis.— Obligate  parasites.  Anaerobic  or  micro- 
aerophilic.  Cells  frequently  elongate  and  fusiform,  staining  some- 
what unevenly.  Filaments  sometimes  formed;  non-branching. 
Non-motile.  No  spores.  Growth  in  laboratory  media  feeble. 
T}^pe  species,  Fusiformis  termitidis  Heelling. 

Genus  4.— Pfeifferella.— Non-motile  rods,  slender,  Gram-nega- 
tive, stain  poorly,  sometimes  forming  threads  and  showing  a  ten- 
dency towrard  branching.  Gelatin  may  be  slowly  liquefied.  Do 
not  ferment  carbohydrates.  Growth  on  potato  characteristically 
honey-like.  Type  species,  Pfeifferella  mallei. 

E.  Order  Eubacteriales.— The  order  Eubacteriales  includes  the 
forms  usually  termed  the  true  bacteria,  that  is,  those  forms  which 
are  considered  least  differentiated  and  least  specialized.  The  cell 
metabolism  is  not  primarily  bound  up  with  hydrogen  sulphid  or 
other  sulphur  compounds,  the  cells  in  consequence  containing  neither 
sulphur  granules  nor  bacteriopurpurin.  The  cells  apparently  do  not 
possess  a  well-organized1  or  well-differentiated  nucleus.  These 
organisms  are  usually  minute  and  spherical,  rod-shaped  or  spiral, 
in  most  genera  not  producing  true  filaments,  and  rarely  branching. 
The  cells  may  occur  singly,  in  chains,  or  other  groupings.  They 
may  be  motile  by  means  of  flagella,  or  non-motile,  but  they  are  never 
notably  flexuous.  Cell  multiplication  occurs  always  by  transverse, 
never  by  longitudinal,  fission.  Some  genera  produce  endospores, 
particularly  the  rod-shaped  types.  Conidia  are  not  observed. 
Chlorophyll  is  absent,  though  the  cells  may  be  pigmented.  The 
cells  may  be  united  into  gelatinous  masses,  but  they  never  form 
motile  pseudoplasmodia  nor  develop  a  highly  specialized  cyst- 
producing  fruiting  stage,  such  as  is  characteristic  of  the  My.ro- 
bacteriales. 

FAMILY  l.—Nitrobacteriacece.— Organisms  usually  rod-shaped 
(sometimes  nearly  spherical  in  Nitrosomonas  and  possibly  in  Azoto- 
bacter).  Cells  motile  or  non-motile.  Branched  involution  forms  in 
Rhizobium  and  Acetobacter.  Endospores  never  formed.  Obligate 
aerobes,  capable  of  securing  growth  energy  by  the  direct  oxidation 
of  carbon,  hydrogen,  or  nitrogen,  or  of  simple  compounds  of  these. 
Non-parasitic  (except  in  Genus  Rhizobium)— usually  water  or  earth 
forms. 

Tribe  1. — Nitrobacterece. — Organisms  deriving  their  life  energy 
from  oxidation  of  simple  compounds  of  carbon  and  nitrogen  (or  of 
alcohol). 

Genus  1.— Hydrogenomonas.— Monotrichic  short  rods  capable  of 
growing  in  the  absence  of  organic  matter  and  securing  growth  energy 
by  the  oxidation  of  hydrogen  (forming  water).  Kaserer  (1905)  who 
first  described  the  organism  states  that  his  species  will  also  grow  well 
on  a  variety  of  organic  substances.  Type  species,  Hydrogenomonas 
pantetropha  (Kaserer  1906)  Orla-Jensen.  Nikleuski  (1910)  described 
two  additional  species,  H.  mtrea  and  H .  flava. 


CLASSIFICATION  OF  BACTERIA  53 

Genus  2.— Methanomottas.— Monotrichic  short  rods  capable  of 
growing  in  the  absence  of  organic  matter  and  securing  growth 
energy  by  the  oxidation  of  methane  (forming  carbon  dioxid  and 
water).  Type  species,  Meth.  methanica. 

Genus  3.— Carboxydomonas.— Autotrophic  rod-shaped  cells  cap- 
able of  securing  growth  energy  by  the  oxidation  of  carbon  monoxid 
(forming  carbon  dioxid).  Type  species,  Carb.  oligocarbophila 
(Beijerinck  and  van  Delden  (1903)  Orla-Jensen  is  described  as  non- 
motile. 

Genus  4.— Acetobactet.— Cells  rod-shaped,  frequently  in  chains, 
non-motile.  Cells  grow  usually  on  the  surface  of  alcoholic  solutions 
as  obligate  aerobes,  securing  growth  energy  by  the  oxidation  of 
alcohol  to  acetic  acid.  Also  capable  of  utilizing  certain  other  carbo- 
naceous compounds,  as  sugar  and  acetic  acid.  Elongated,  filament- 
ous, club-shaped,  swollen,  and  even-branched  cells  may  occur  as 
involution  forms.  Type  species,  Ace.  aceti. 

Genus  5.—  Nitrosomonas.— Cells  rod-shaped  or  spherical,  motile, 
or  non-motile,  if  motile  with  polar  flagella.  Capable  of  securing 
growth  energy  by  the  oxidation  of  ammonia  to  nitrites.  Growth  on 
media  containing  organic  substances  scanty  or  absent.  Type  species, 
Nitro.  europoea  Winogradsky. 

Genus  6.— Nitrobacter.— Cells  rod-shaped,  non-motile,  not  grow- 
ing readily  on  organic  media  or  in  the  presence  of  ammonia.  Cells 
capable  of  securing  growth  energy  by  the  oxidation  of  nitrites  to 
nitrates.  Type  species,  Nitro.  Winogradskyi. 

Tribe  2. — Azotobacterece  (Nitrogen-fixing  organisms). 

Genus  7.— Azotobacter.—  Relatively  large  rods,  or  even  cocci, 
sometimes  almost  yeast-like  in  appearance,  dependent  primarily 
for  growth  energy  upon  the  oxidation  of  carbohydrates.  Motile  or 
non-motile;  when  motile,  with  tuft  of  polar  flagella.  Obligate 
aerobes  usually  growing  in  a  film  upon  the  surface  of  the  culture 
medium.  Capable  of  fixing  atmospheric  nitrogen  when  grown  in 
solutions  containing  carbohydrates  and  deficient  in  combined  nitro- 
gen. Type  species,  Azotobacter  chroococcum  Beijerinck. 

Genus  8.  —  Rhizobium.— Minute  rods,  motile  when  young.  Invo- 
lution forms  abundant  and  characteristic  when  grown  under  suitable 
conditions.  Obligate  aerobes,  capable  of  fixing  atmospheric  nitrogen 
when  grown  in  the  presence  of  carbohydrates  in  the  absence  of 
compounds  of  nitrogen.  Produce  nodules  upon  the  roots  of  legu- 
minous plants.  Type  species,  R.  leguminosarum  Frank. 

FAMILY  II.— Pseudomonada'cece.—  Rod-shaped,  short,  usually 
motile  by  means  of  polar  flagella  or  rarely  non-motile.  Aerobic  and 
facultative.  Frequently  gelatin  liquefiers  and  active  ammonifiers. 
No  endospores.  Gram  stain  variable  though  usually  negative. 
Fermentation  of  carbohydrates  as  a  rule  not  active.  Frequently 
produces  a  water-soluble  pigment  which  diffuses  through  the  medium 


54  CLASSIFICATION  OF  BACTERIA 

as  green,  blue,  purple,  brown,  etc.  In  some  cases  a  non-diffusible 
yellow  pigment  is  formed.  Many  yellow  species  are  plant  parasites. 

Genus  l.  —  Pseudomonas.  —  Characters,  those  of  family.  Type 
species,  Ps.  aeruginosa  (Schroeter)  Frost? 

FAMILY  III.— Spirillacece.— Cells  elongate,  more  or  less  spirally 
curved.  Cell  division  always  transverse,  never  longitudinal.  Cells 
non-flexuous.  Usually  without  endospores.  As  a  rule  motile  by 
means  of  polar  flagella,  sometimes  non-motile.  Typically  water 
forms,  though  some  species  are  intestinal  parasites. 

Genus  1.— Vibrio.— Cells  short  bent  rods,  rigid,  single,  or  united 
into  spirals.  Motile  by  means  of  a  single  (rarely  two  or  three)  polar 
flagellum  which  is  usually  relatively  short.  Many  species  liquefy 
gelatin  and  are  active  ammonifiers.  Aerobic  and  anaerobic.  No 
endospores.  Usually  Gram-negative.  Water  forms,  a  few  parasites. 
Type  species,  V.  comma  (Koch  1884)  Schroeter  1886. 

Genus  2.— Spirillum.— Cells,  rigid  rods  of  various  thicknesses, 
length,  and  pitch  of  the  spiral,  forming  either  long  screws  or  portions 
of  a  turn.  Usually  motile  by  means  of  a  tuft  of  polar  flagella  (5 
to  20)  which  are  mostly  half  circular,  rarely  wavy-bent.  These 
flagella  occur  on  one  or  both  poles;  their  number  varies  greatly  and 
difficult  to  determine;  since  in  stained  preparations  several  are 
often  united  into  a  common  strand.  Endospore  formation  has  been 
reported  in  some  species.  Habitat :  water  or  putrid  infusions.  Type 
species,  S.  undula  (Mueller  1786)  Ehrenberg. 

FAMILY  IV.  —  Coccacece.— Cells  in  their  free  conditions,  spherical; 
during  division  somewhat  elliptical.  Division  in  one,  two,  or  three 
planes.  If  the  cells  remain  in  contact  after  division  they  are  fre- 
quently flattened  in  the  plane  of  division  and  form  chains,  packets, 
or  irregular  masses.  Motility  rare.  Endospores  absent.  Metab- 
olism complex,  usually  involving  the  utilization  of  amino-acids 
or  carbohydrates.  Pigment  often  produced. 

Tribe  A.— Neisserece.— Strict  parasites,  failing  to  grow  or  growing 
very  poorly  on  artificial  media.  Cells  normally  in  pairs.  Gram- 
negative.  Growth  fairly  abundant  on  serum  media. 

Genus  l.—Neisseria.— Characters,  those  of  tribe.  Type  species, 
N.  gonorrhoece  Trevisan. 

Tribe  B.—Streptococcece.— Parasites  (thriving  only  or  best  on 
or  in  the  animal  body)  except  genus  Leuconostoc.  Grow  well  under 
anaerobic  conditions.  Many  forms  grow  with  difficulty  on  serum- 
free  media,  none  very  abundantly.  Planes  of  fission  usually  parallel 
producing  pairs,  or  short,  or  long  chains,  never  packets.  Generally 
stain  by  Gram.  Produce  acid  but  no  gas  in  glucose  and  generally  in 
lactose  broth.  Pigment,  if  any,  white  or  orange. 

Genus  2.—  Diplococcus.— Parasites,  growing  poorly,  or  not  at  all, 
on  artificial  media.  Cells  usually  in  pairs  of  somewhat  elongated 
cells,  often  capsulated,  sometimes  in  chains.  Gram-positive.  Fer- 


CLASSIFICATION  OF  BACTERIA  55 

mentative  powers  high,  most  strains  forming  acid  in  glucose,  lactose, 
sucrose,  and  inulin.  Type  species,  D.  pneumonice  Weichselbaum. 

Genus  3. — Leuconostoc. — Saprophytes  usually  growing  in  cane 
sugar  solutions.  Cells  in  chains  or  pairs  united  in  large  zoogleal 
masses.  Some  types  at  least  Gram-negative.  Type  species  L. 
m  e  s  enter  old  es  (Cienkowski)  van  Tieghem. 

Genus  4. — Streptococcus. — Chiefly  parasites.  Cells  normally  in 
short  or  long  chains  (under  unfavorable  conditions  sometimes  in 
pairs  and  small  groups,  never  in,  large  packets) .  Generally  stain  by 
Gram.  Capsules  rarely  present,  no  zoogleal  masses.  On  agar 
streak,  efl'used  translucent  growth  often  with  isolated  colonies.  In 
stab  culture  little  surface  growth.  Many  sugars  fermented  with 
formation  of  large  amount  of  acid,  but  inulin  is  rarely  attacked. 
Generally  fail  to  liquefy  gelatin  or  reduce  nitrates.  Type  species, 
S.  pyogenes  Rosenbach. 

Genus  5.— Staphylococcus.— Parasites.  Cells  in  groups  and  short 
chains,  very  rarely  in  packets.  Generally  stain  by  Gram.  On  agar 
streak  good  growth,  of  white  or  orange  color.  Glucose,  maltose, 
sucrose,  and  often  lactose,  fermented  with  formation  of  moderate 
amount  of  acid.  Gelatin  often  liquefied  very  actively.  Type  species, 
S.  aureus  Rosenbach. 

Tribe  C.—Micrococcece.— Facultative  parasites  or  saprophytes. 
Thrive  best  under  aerobic  conditions.  Grow  well  on  artificial  media, 
producing  abundant  surface  growths.  Planes  of  fission  often  at 
right  angles;  cell  aggregates  in  groups,  packets,  or  zoogleal  masses. 
Generally  decolorize  by  Gram.  Pigment  yellow  or  red. 

Genus  6.— Micrococcus.— Facultative  parasites  or  saprophytes. 
Cells  in  plates  or  irregular  masses  (never  in  long  chains  or  packets). 
Generally  decolorize  by  Gram.  Growth  on  agar  abundant,  with 
formation  of  yellow  pigment.  Glucose  broth  slightly  acid,  lactose 
broth  generally  neutral.  Oelatin  frequently  liquefied,  but  not 
rapidly.  Type  species,  M.  luteus  (Schroeter)  1872b,  Cohn. 

Genus  7.—Sarcina.—Sarcina  differs  from  Micrococcus  solely  in 
the  fact  that  cell  division  occurs  under  favorable  conditions  in  three 
planes,  forming  regular  packets.  Type  species,  Sarcina  ventriculi 
Goodsir. 

Genus  8. —  Rhodococcus. — Saprophytes.  Cells  in  groups  or  regular 
packets.  Generally  decolorize  by  Gram.  Growth  on  agar  abundant 
with  formation  of  red  pigment.  Glucose  broth  slightly  acid,  lactose 
broth  neutral.  Gelatin  rarely  liquefied.  Nitrates  generally  reduced. 
Type  species,  R.  rhodochrous  Zopf . 

FAMILY  V.—Bacteriacece.— Rod-shaped  cells  without  endospores. 
Usually  Gram-negative.  Flagella  when  present  peri trichic.  Metab- 
olism complex,  amino-acids  being  utilized  and  generally  carbo- 
hydrates. 


56  CLASSIFICATION  OF  BACTERIA 

Tribe  l.—Chromobactercp.— Water  bacteria  producing  a  red  or 
violet  pigment. 

Genus  l.  —  Erythrobacilhis.— Small  aerobic  bacteria,  producing  a 
red  or  pink  pigment,  usually  a  lipochrome.  Gram  stain  variable. 
It  is  possible  that  related  yellow  and  orange  chromogens  should  be 
included  here  as  well.  Type  species,  E.  prodigiosus  (Ehrenberg). 

Genus  2.— Chromobacterium.— Aerobic  bacteria,  producing  a  violet 
chromoparous  pigment,  soluble  in  alcohol  but  not  in  chloroform. 
Motility  and  Gram  reaction  variable.  Type  species,  Chr.  mokecum 
Bergonzini. 

Tribe  2.— Erwinece.— Plant  pathogens.  Growth  usually  whitish, 
often  slimy.  Indol  generally  not  produced.  Acid  usually  formed  in 
certain  carbohydrate  media,  but  as  a  rule  no  gas. 

Genus  3.— Erwinia.— Characters  those  of  the  tribe.  Type  species, 
E.  amylowra. 

Tribe  3.—  Zopfece.— Gram-positive  rods,  growing  freely  on  arti- 
ficial media.  Not  attacking  carbohydrates. 

Genus  4.— Zopfius.—  Long  rods  occurring  in  evenly  curved  chains. 
Gram-positive.  Motile.  Proteus-like  growth  on  media.  Faculta- 
tive anaerobes.  Carbohydrates  and  gelatin  not  attacked,  hydrogen 
sulphid  not  formed.  Type  species,  Z.  zopfi  (Kurth)  Wenner  and 
Rettger. 

Tribe  4.— Bacterece.— Gram-negative  rods  growing  freely  on 
artificial  media.  Generally  forming  acid  from  carbohydrates  and 
often  gas  composed  of  CO2  and  H2. 

Genus  5. — Proteus. — Highly  pleomorphic  rods,  filaments  and 
curved  cells  being  common  as  involution  forms.  Gram-negative. 
Actively  motile.  Characteristic  ameboid  colonies  on  moist  media. 
Liquefy  gelatin  rapidly  and  produce  vigorous  decomposition  of 
proteins.  Ferment  glucose  and  sucrose  (but  usually  not  lactose) 
with  formation  of  acid  and  gas  (the  latter  being  C02  only).  Type 
species,  P.  vulgaris  Hauser. 

Genus  6.— Bacterium.— Gram-negative,  evenly  staining  rods. 
Often  motile,  with  peritrichic  flagella.  Easily  cultivable,  forming 
grape-vine  leaf  or  convex  whitish  surface  colonies.  Liquefy  gelatin 
rarely.  All  forms  except  B.  alcaligenes  and  the  B.  abortus  group 
attack  the  hexoses  and  most  species  ferment  a  large  series  of  carbo- 
hydrates. Acid  formed  by  all,  gas  (CO2  and  H2)  only  by  one  series. 
Typically  intestinal  parasites  of  man  and  the  higher  animals  although 
several  species  may  occur  on  plants,  and  one  (B.  aerogenes)  is  widely 
distributed  in  nature.  Many  species  pathogenic.  Type  species, 
B.  coli  Escherich. 

Tribe  5.—Lactobacillece.—l\ods  often  long  and  slender,  Gram- 
positive,  non-motile,  without  endospores.  Usually  produce  acid 
from  carbohydrates,  as  a  rule  lactic.  When  gas  is  formed  it  is  CO2 


CLASSIFICATION  OF  BACTERIA  57 

without  H2.  The  organisms  are  usually  somewhat  thermophilic. 
As  a  rule  micro-aerophilic;  surface  growth  on  media  poor. 

Genus  7. — Lactobacillus . — Generic  characters  those  of  the  tribe. 
Type  species,  L.  caucasicus  (Kern?)  Beijerinck. 

Tribe  6. — Pasteur ellece. — Gram-negative  rods,  showing  bipolar 
staining.  Parasitic  forms  of  slight  fermentative  power. 

Genus  8.— Pasteurella.— Aerobic  and  facultative.  Powers  of  car- 
bohydrate fermentation  slight;  no  gas  produced.  Gelatin  not 
liquefied.  Parasitic,  frequently  pathogenic,  producing  plague  in 
man  and  hemorrhagic  septicemia  in  the  lower  animals.  Type 
species,  P.  cholerse-gallinarum  (Fliigge)  1886  Trevisan. 

Tribe  7.— Hemophilcece.— Minute  parasitic  forms  growing  only  in 
presence  of  hemoglobin,  ascitic  fluid  or  other  body  fluids. 

Genus  9.— Hemophilus.— Minute  rod-shaped  cells,  sometimes 
thread  forming  and  pleomorphic,  non-motile,  without  spores,  strict 
parasites,  growing  best  (or  only)  in  presence  of  hemoglobin,  and  in 
general  requiring  blood  serum  or  ascitic  fluid.  Gram-negative. 
Type  species  //.  influenzas  (Pfeiffer  1893). 

FAMILY  VII.—  Bacillacece.— Rods  producing  endospores,  usually 
Gram-positive.  Flagella  when  present  peritrichic.  Often  decom- 
pose protein  media  actively  through  agency  of  enzymes. 

Genus  1.— Bacillus.— Aerobic  forms.  Mostly  saprophytes. 
Liquefy  gelatin.  Often  occur  in  long  threads  and  form  rhizoid 
colonies.  Form  of  rod  usually  not  greatly  changed  at  sporulation. 
Type  species,  B.  subtilis  Cohn. 

Genus  2.— Clostridium.— Anaerobes  or  micro-aerophiles.  Often 
parasitic.  Rods  frequently  enlarged  at  sporulation,  producing 
clostridium  or  pleotridium  forms.  Type  species,  C.  butyricum 
Prazmowski. 


CHAPTER  V. 
COMPOSITION  OF  BACTERIA. 

THE  elementary  composition  of  bacteria  is  the  same  as  that 
of  the  higher  plants.  This  is  also  true  concerning  the  main  chem- 
ical constituents  composing  their  body.  But  the  proportions  of 
these  latter  at  times  vary  quite  widely.  Moreover,  some  micro- 
organisms contain  constituents  not  found  in  higher  plants. 

Elementary  Composition.— Bacteria  on  analysis  yield  carbon,  hy- 
drogen, oxygen,  nitrogen,  potassium,  phosphorus,  sulphur,  calcium, 
magnesium,  iron,  aluminum  and  manganese.  As  to  whether  the 
last  two  are  essential  to  normal  development  is  not  certain.  In 
some  species  they  are  known  to  be  non-essential,  whereas  in  others, 
for  instance  the  Azotobacter,  they  seem  to  play  an  important  part. 

Moisture. —Moisture  is  essential  for  all  plant  and  animal  life  and 
is  always  abundant  in  actively  growing  cells;  hence,  we  expect, 
and  do  actually  find,  large  quantities  of  water  in  bacteria.  The 
quantity  present  in  the  actively  growing  cell  varies  from  as  low  as 
70  per  cent,  to  as  high  as  90  per  cent.  Generally  speaking,  young 
cultures  contain  less  moisture  than  do  older  cultures;  this  appears 
to  be  true  until  the  spore  stage  is  reached,  after  which  the  quantity 
of  water  greatly  decreases.  The  temperature  at  which  the  cultures 
are  grown  also  governs  in  a  measure  the  quantity  of  water  present, 
this  being  less  when  grown  at  37°  C.  than  when  grown  at  20°  C. 
The  cultural  media  undoubtedly  play  a  great  part  in  determining 
the  moisture  content  of  the  cells.  It  is  probably  rather  low  in 
bacteria  obtained  from  alkali  soil  or  from  saline  waters. 

Organic  Constituents.— The  bacterial  cell  contains  carbohydrate- 
like  bodies,  proteins,  extractives  (fats,  fatty  acids,  waxes  and 
lipoids),  and  enzymes.  In  addition  to  these  some  bacteria  also 
contain  pigments,  toxins  and  possibly  ptomains.  The  quantity 
and  quality  of  each,  especially  of  the  last  four,  vary  greatly  with 
the  class  of  organisms  and  the  conditions  under  which  they  are 
grown. 

Carbohydrates  are  really  conspicuous  by  their  absence  in  most 
bacterial  cells,  but  the  following  members  of  the  carbohydrate 
group  have  been  recognized  in  varying  quantities  in  some  bacteria: 
cellulose,  hemicellulose,  dextrin,  starch,  glycogen  and  a  number  of 
the  sugars. 

Extractives,  although  found  to  a  limited  extent  in  all  microorgan- 
isms, are  found  in  larger  quantities  in  the  tubercle  bacillus  and  other 


ORGANIC  CONSTITUENTS  59 

members  of  the  acid-fast  group.  In  some  members  of  this  group 
the  extractives  vary  from  20  to  40  per  cent,  of  the  total  dry  residue. 
In  the  early  studies  of  the  chemistry  of  bacterial  cells  it  was  assumed 
that  the  alcoholic  and  ethereal  extracts  consisted  of  fats  exclusively. 
Tributyrin,  tripalmatin,  tristearin,  triolein,  lecithin  and  various 
waxes  have  been  recognized. 

Klebs  found  in  the  tubercle  bacillus  20.5  per  cent,  of  a  red  fat 
melting  at  42°  and  1.14  per  cent,  of  a  white  fat  melting  above  50°, 
the  latter  being  insoluble  in  ether  but  soluble  in  benzol.  De  Schwei- 
nitz  and  Dorset  concluded  that  the  fat  of  the  tubercle  bacillus  con- 
tains palmitic  and  arachidic  acids,  while  that  of  the  glanders  bacillus 
contains  oleic  and  palmitic.  They  also  obtained  a  crystalline  acid, 
for  which  they  suggested  the  name  tuberculinic  acid.  This  is  quite 
different  from  Ruppel's  nucleic  acid.  It  had  an  elementary  com- 
position of  C7Hi0O4.  The  authors  called  attention  to  the  similarity 
in  composition  and  properties  of  this  body  and  tetraconic  acid. 
They  suggest  that  it  may  be  the  substance  which  is  responsible 
for  the  coagulating  necrosis  and  reduction  in  temperature. 

Kresslig  extracted  tubercle  bacilli  successfully  with  ether,  chloro- 
form, benzol  and  alcohol,  and  obtained  38.95  per  cent,  of  fatty  and 
waxy  substances.  Repeated  extraction  with  chloroform  gave  a 
dark  brown  mass  of  the  consistency  and  color  of  beeswax  and  melting 
at  46°.  He  found  14.38  per  cent,  of  free  fatty  acid,  77.25  per  cent, 
of  neutral  fat  and  esters  of  fatty  acids,  and  some  volatile  fatty 
acid,  probably  butyric.  He  concluded  that  the  fat  of  the  tubercle 
bacillus  is  quite  different  from  that  obtained  from  any  other  source. 

The  fats  and  waxes  are  probably  both  intra-  and  extracellular, 
for  extraction  of  the  intact  cell  yields  some  and  the  crushed  cell 
yields  still  more.  The  quantity  found  within  the  cell  varies  greatly, 
depending  on  the  media  upon  which  the  organism  is  grown.  Meyer 
found  that  the  fat  in  Bacillus  tumescens  gradually  increases  until 
spore  formation  occurs,  when  it  disappears;  the  spores  are  also  free 
from  fat.  This,  however,  is  not  general  for  the  spores  of  some 
organisms  contain  proportionally  more  fat  than  do  the  vegetative 
forms. 

Proteins.— The  bulk  of  the  dry  matter  of  the  bacterial  cell  is  com- 
posed of  proteins.  The  following  analysis  reported  by  Iluppel 
indicates  the  composition  of  the  tubercle  cell: 

Nucleic  (tuberculinic  acid)       ........        8.5  per  cent. 

Nucleoprotamin 25.5 

Nucleoproteid 23.0 

Albuminoids * .        8.3 

Fat  and  wax 26.5 

Ash    .      . 9.2 

The  wonderful  synthetic  reaction  catalyzed  by  the  Azotobacter  cell 
has  directed  the  attention  of  workers  to  this  specific  organism. 


60  COMPOSITION  OF  BACTERIA 

Therefore,  our  knowledge  of  the  composition  of  this  organism  is 
probably  more  nearly  complete  than  it  is  of  many  other  species. 

Berthelot  early  recognized  that  the  nitrogen  fixed  by  the  Azoto- 
bacter  is  insoluble  in  water,  thus  indicating  its  protein  nature. 
Lipman  found  there  was  a  small  but  appreciable  quantity  of  nitro- 
gen in  both  young  and  old  cultures  of  A.  mnelandii  not  precipi- 
tated by  lead  acetate  and  a  large  proportion  not  precipitated  by 
phosphotungstic  or  tannic  acid.  Further  work  indicated  that  the 
substance  was  either  amino-acids  or  comparatively  simple  peptids. 
He  considered  that  one  of  the  early  substances  synthesized  by  these 
organisms  was  alanin.  An  analysis  of  the  Azotobacter  membrane 
gave  the  following: 

Nitrogen  as  ammonia 0.98  per  cent. 

Basic  nitrogen 2.76          " 

Non-basic  nitrogen 6.39         " 

Nitrogen  in  MgO  precipitate 0.42         " 

Total  nitrogen    .      .  .  10.45          " 


This,  he  finds,  corresponds  remarkably  closely  to  legumin.  That 
it  is  complex  is  indicated  by  the  fact  that  it  is  not  readily  assimilated 
by  plants. 

Stoklasa  found  the  Azotobacter  cell  to  contain  10.2  per  cent,  of 
total  nitrogen  and  8.6  per  cent,  of  ash.  The  ash  was  from  58  to 
62.35  per  cent,  phosphoric  acid.  The  nitrogen  and  phosphorus 
were  mainly  in  the  forms  of  nucleoproteins  and  lecithin.  The 
percentage  of  both  nitrogen  and  phosphorus  in  the  cell  increases 
with  age. 

The  most  complete  analysis  of  the  Azotobacter  cells,  so  far 
reported  shows  them  to  contain,  when  grown  on  dextrin  agar  and 
rapidly  dried  at  30°  C.,  12.92  per  cent,  of  protein.  The  protein  is 
similar  to  other  plant  proteins.  It  contains  10  per  cent,  of  ammonia 
nitrogen,  26.5  per  cent,  of  diamino-nitrogen,  and  60  per  cent,  of 
mono-amino-nitrogen.  It  contains  the  amino-acids  normally  found 
in  proteins  but  the  quantity  of  lysin  present  is  high,  whereas  the 
histidin  is  present  only  in  traces. 

An  examination  made  by  Nishimura  of  a  pure  culture  of  a  water 
bacillus  gave  the  following  as  the  composition  of  the  dry  matter  in 
the  bacillus. 

Albumin 63.50  per  cent. 

Carbohydrates 12.2 

Alcohol  extract 3.2 

Ether  extract 5.10 

Ash 11.20 

Lecithin 0.68 

Xanthin 0.17 

Guanin 0.14 

Adenin .  0.08 


VARIATION  IN  DIFFERENT  PARTS  OF  THE  CELL        61 

The  organism  is,  therefore,  extremely  rich  in  protein,  and, 
although  the  albumin  predominates  it  is  not  free  from  nucleoprotein, 
as  is  seen  from  the  presence  of  the  purine  bases,  xanthin,  guanin 
and  adenin. 

Inorganic  Constituents.— The  ash-content  of  the  bacterial  cell  is 
not  far  different  qualitatively  from  that  of  the  higher  plants. 
Quantitatively,  however,  there  is  a  marked  difference,  the  ash- 
content  of  bacteria  being  comparatively  high.  The  ash-content 
of  the  cell  is  subject  to  wide  variation,  depending  on  the  specific 
organisms  and  especially  on  the  media  upon  which  it  is  grown. 
This  may  be  seen  from  the  following  results  by  Cramer  who  grew 
the  Cholera  vibrio  on  various  media. 


Composition  of  medium  in 
which  organisms  were 
grown. 

1  per  cent,  soda 
bouillon  (regular 
broth  +  1  oer  cent. 
NaOH). 

Phosphate 
bouillon  (regular 
broth  +  1  percent, 
sod.  phosphate). 

NaCl  bouillon 
(regular  broth  -f 
3  per  cent.  NaCl). 

Ash-content  of  bacteria  in  dry  sub- 

stance     

9.30 

22.30 

25.90 

Ash-content  of  moist  mass 

1.34 

2.75 

3.73 

Ash-content   of  medium  in   moist 

mass      

1.25 

2.50 

4.12 

Phosphoric  acid  in  bacterial  ash   . 

28.70 

34.80 

10.90 

Phosphoric  acid  in  media  ash    . 

7.90 

39.80 

2.10 

Chlorin  in  bacterial  ash 

16.90 

7.97 

40.70 

Chlorin  in  media  ash      .... 

23.00 

11.40 

49.20 

Analyses  have  been  reported  in  which  the  phosphoric  acid-con- 
tent reaches  as  high  as  one-half  the  total  ash-content  of  the  cell. 
It  is  quite  probable  that  a  great  proportion  of  this  is  combined  with 
the  nucleic  acid  in  the  nucleoproteins. 

Variation  in  Composition  of  Different  Parts  of  the  Cell.— As  has 
been  pointed  out,  the  bacterial  cell  is  not  homogeneous  but  is  made 
up  of  fairly  distinct  parts,  namely,  ectoplasm,  capsule  and  cyto- 
plasm and  nuclear  material.  These  constituents  vary  noticeably 
in  their  chemical  composition.  Although  the  ectoplasm  at  times 
contains  in  some  species  of  bacteria  small  quantities  of  cellulose 
and  hemicellulose,  yet  the  predominating  substance  is  chitin,  a 
substance  which  may  be  considered  as  an  intermediary  compound 
between  the  carbohydrates  and  proteins.  When  pure,  chitin  yields 
over  80  per  cent,  of  its  weight  as  glucosamine.  It  yields  first  acetic 
acid  and  chitosan: 


Ci8H3oN2O12 
Chitin. 


2H20    =   2CH3COOH 
Acetic  acid. 


Chitosan. 


Chitosan  on  further  hydrolysis  yields  acetic  acid  and  glucosamine: 


Ci4H26N2O10 
Chitosan. 


2H20    =  CHsCOOH 
Acetic  acid. 


Glycosamine. 


62  COMPOSITION  OF  BACTERIA 

The  cell  membrane  is,  therefore,  more  nearly  that  of  the  animal 
than  the  plant.  The  brown  color  obtained  on  staining  some 
bacteria  with  iodin  has  led  observers  to  believe  that  they  contain 
glycogen,  whereas  the  blue  color  with  the  same  reagent  is  attributed 
to  starch. 

The  capsules  contain  comparatively  large  quantities  of  mucin. 
These  are  protein-like  substances  which  may  be  precipitated  by 
alcohol.  They  give  most  of  the  protein  reactions  and,  in  addition, 
when  heated  with  an  acid,  acquire  the  property  of  reducing  Fehling's 
solution,  thus  showing  them  to  contain  a  carbohydrate  complex  in 
addition  to  the  protein. 

The  cytoplasm  consists  largely  of  bacterial  proteins  which  appear 
to  be  specific  in  character  for  any  given  species.  Within  this  are 
large  quantities  of  the  nucleoproteins,  for  on  hydrolysis  large 
quantities  of  the  purine  bases  are  obtained.  Vaughan,  Wheeler 
and  Leach  conclude  that  the  bacterial  cytoplasm  contains  carbo- 
hydrates, nuclein  bodies,  and  polymers  of  mono-  and  di-amino-acids. 
They  are  glyconucleoproteins.  Spores  differ  from  the  vegetative 
organism  in  that  they  contain  but  small  quantities  of  water. 

REFERENCES. 

Vaughan:     Protein-split  Products  in  Relation  to  Immunity  and  Disease. 
Kendall:     Bacteriology — General,  Pathological,  Intestinal. 
Kruse:     Allgemeine  Micro  biologic. 


CHAPTER  VI. 
FOOD  REQUIREMENTS. 

FOOD  is  any  substance  which  bacteria  can  utilize  in  obtaining 
either  building  material  or  energy  for  the  cell  activity.  The  quan- 
tity and  quality  of  food  necessary  vary  widely  with  the  different 
species.  However,  all  foods  must  contain  certain  essential  ele- 
ments. Our  knowledge  at  the  present  time  indicates  these  ele- 
ments to  be  carbon,  hydrogen,  oxygen,  phosphorus,  potassium, 
nitrogen,  sulphur,  calcium,  iron  and  magnesium,  or,  using  the  key 
for  remembering  as  suggested  by  Dr.  Hopkins,  we  have  C.  Hopk'ns 
CaFe  Mg "C.  Hopk'ns  Cafe  -  -  mighty  good." 

Minimum  Requirements. — In  considering  the  food  used  by  bacteria 
a  minimum  and  a  maximum  requirement  must  be  recognized. 
These  two  extremes  differ  greatly,  for  although  the  minimum  quan- 
tities appear  inconceivably  small  the  maximum  ones  are  enormous. 
One  may  obtain  a  fair  idea  of  the  minimum  requirements  from  the 
following  calculation  made  by  Rahn:  "The  quantity  of  organic 
and  inorganic  matter  just  sufficient  to  support  a  very  weak  growth 
is  certainly  very  small,  since  a  few  species  will  multiply  to  some 
extent  in  ordinary  distilled  water.  Such  water,  after  having  stood 
for  some  time,  is  found  to  contain  several  thousand  bacteria  per 
cubic  centimeter.  It  may  seem  to  the  laymen  that  in  such  water 
it  would  be  possible  to  detect  easily  the  organic  and  inorganic  matter 
of  the  microorganisms  so  that  it  could  not  be  considered  distilled 
water.  An  estimate  of  the  weight  of  bacteria  demonstrates,  how- 
ever, that  this  is  not  the  case.  If  we  suppose  the  average  bacterial 
cell  to  be  a  cylinder  whose  base  measures  1  square  micron  and 
whose  height  is  2  microns  (which  is  a  high  estimate) .  The  volume 
of  such  a  cell  would  be  1  X,l  X  2  cubic  microns  =  0.001  X  0.001 
X  0.002  mm.  =  0.000,000,002  cu.mm.  The  specific  gravity  of 
bacteria  being  very  nearly  one,  the  weight  of  one  bacterium  would 
be  0.000,000,002  mg.  One  hundred  thousand  cells  per  cubic  centi- 
meter means  100,000,000  cells  per  liter,  which  would  weight  0.2 
mg.  Of  this  total  weight,  at  least  four-fifths  is  water  and  only  one- 
fifth  is  solid  matter.  The  total  solid  matter  in  1  liter  of  water 
containing  100,000  bacteria  per  cubic  centimeter  amounts  to  the 
immeasurable  quantity  of  0.04  mg.  Such  water  will  pass  the  test 
for  distilled  water.  How  much  food  the  bacteria  in  distilled  water 
have  used  is  impossible  to  say,  since,  besides  the  traces  of  minerals 


64  FOOD  REQ UIREMENTS 

in  the  water,  they  obtain  some  food  from  volatile  compounds  of 
the  air,  like  carbon  monoxid  (CO),  carbon  dioxid  (COz),  ammonia 
(NH8),  hydrogen  (H)-and  perhaps  methane  (CH4).  Under  all 
circumstances  the  amount  of  food  used  is  very  small." 

Maximum  Requirements.— The  maximum  quantity  of  food  which 
may  be  decomposed  by  bacteria  is  often  enormous.  They  quickly 
decompose  the  body  of  an  ox  after  its  death.  Tons  of  material 
run  into  the  septic  tanks  of  large  cities,  all  of  which  is  rapidly 
decomposed  by  bacteria.  It  is,  however,  usually  the  case  that  the 
speed  of  the  reaction  is  great  at  first,  but  soon  slows  up  or  comes 
to  a  complete  stop.  This  is  due  to  the  fact  that  the  accumulation 
of  end-products  interferes  with  the  growth  of  bacteria.  This  is 
true  in  milk  where  at  first  the  lactose  is  rapidly  changed  to  lactic 
acid,  which  if  not  neutralized  soon  becomes  concentrated  enough 
to  slow  up  the  reaction.  This  is  also  true  with  the  changes  going 
on  in  sauerkraut  and  silage. 

Function  of  the  Food.— The  food  utilized  by  bacteria  has  two 
functions,  namely,  the  furnishing  of  energy  and  the  acting  as  cellular 
building  material.  The  quantity  required  by  each  bacterial  cell 
for  building  material  is  not  great,  for  MacNeal  and  his  associates 
found  that  the  dry  matter  of  550,000,000  cells  of  B.  coli  weigh 
only  0.1  mg.  Others  have  estimated  the  weight  of  a  single  colon 
bacillus  to  be  0.000,000,163  mg.,  or  it  would  require  1,600,000,000 
colon  bacilli  to  weight  approximately  1  mg.  The  waste  products 
and  repair  material  would  make  the  cellular  requirements  slightly 
greater  than  this,  but  from  these  figures  it  is  evident  that  the  actual 
quantity  required  by  a  cell  for  building  material  is  extremely  small. 
Even  this,  however,  is  not  immaterial,  for  Conn  starting  with  the 
assumption  that  the  period  of  generation  is  a  half  hour  makes  the 
following  calculation.  "If  we  take  a  single  bacillus  measuring 
2M  in  length  and  I/*  in  breadth,  with  a  weight  of  0.000,000,001,571 
mg.,  it  will  increase,  according  to  the  aforesaid  assumption,  at 
such  a  rate  that  in  two  days'  time  its  progeny  will  amount  to 
281,000,000,000,  and  will  occupy  a  volume  equal  to  about  J  liter 
(30.51  cu.  in.);  within  a  further  three  days  the  quantity  would 
increase  to  a  mass  sufficient  completely  to  fill  the  beds  of  all  the 
oceans  of  the  globe."  Due  to  the  accumulation  of  waste  products 
they  never  continue  to  multiply  long  at  such  a  rate,  but  the  numbers 
in  suitable  media  often  become  hundreds  of  millions  per  cubic 
centimeter  before  retardation  occurs. 

Source  of  Energy.— Animals  and  plants  require  energy  in  their 
life  activity,  the  former  obtaining  it  directly  from  the  kinetic 
energy  of  the  sun  which  they  store  up  as  potential  energy. 
This  is  liberated  by  the  animal  in  the  process  of  oxidation.  Now, 
bacteria  do  not  possess  the  powers  of  the  higher  plants  to  utilize 
directly  the  energy  of  the  sun,  but,  like  the  animals  they  are 


MOISTURE  65 

dependent  on  the  stored  energy  of  the  plant  and  animal  kingdom. 
From  their  method  of  oxidation  it  is  necessary  to  recognize  two 
groups  of  bacteria:  (1)  Those  which  completely  oxidize  their 
food,  the  carbon  and  hydrogen  occurring  in  the  final  products  as 
carbon  dioxid  and  water;  (2)  those  which  only  partly  decompose 
their  food,  thus  leaving  much  of  the  energy  still  within  the  food. 
Now  the  actual  food  requirements  of  the  two  classes  of  organism  for 
the  accomplishment  of  the  same  end,  in  so  far  as  energy  is  con- 
cerned, is. materially  different.  For  instance,  the' complete  oxida- 
tion of  glucose  to  carbon  dioxid  and  water  as  brought  about  by  some 
yeasts  according  to  the  equation  C6Hi2O6  +  6O2  =  6CO2  +  6H2O  + 
674  cal. ;  whereas,  when  only  partly  oxidized  to  alcohol  it  would  be 
C6H12O6  =  2C2H5OH  +  2CO2  +  22  cal. 

The  energy  obtained  in  the  first  case  is  over  thirty  times  that 
obtained  in  the  second,  and  the  quantity  of  food  decomposed  would 
be  relatively  greater  in  the  latter  than  in  the  former.  It  has  been 
estimated  that  the  lactic  acid  bacteria  decompose  their  own  weight 
of  sugar  in  one  hour. 

Although  all  organisms  require  the  elements  listed  at  the  begin- 
ning of  this  chapter,  yet  the  nature  of  the  organic  compound  required 
varies  greatly  with  different  species. 

Moisture.— Moisture  may  be  considered  the  most  important 
factor  of  life.  "It  is  little  short  of  astounding  that  living  matter 
with  all  its  wonderful  properties  of  growth,  movement,  memory, 
intelligence,  devotion,  suffering  and  happiness  should  be  composed 
to  the  extent  of  from  70  to  90  per  cent,  of  nothing  more  complex  or 
mysterious  than  water.  Such  a  fact  as  this  is  most  perplexing,  espe- 
cially when  all  experiments  show  that  this  water  is  playing  a  pro- 
foundly important  part  in  the  generation  of  the  vital  phenomena. 
Any  interference  with  the  amount  normally  present  makes  a  change 
at  once  in  the  activities  of  the  cell.  In  fact  we  might  say  that 
'all  living  matter  lives  in  water/  as  Claude  Bernard  put  it.  For 
not  only  is  this  obviously  true  in  the  lower  and  simpler  forms  of 
animals  and  plants,  which  are  little  more  than  naked  masses  of 
protoplasm  living  in  water,  but  it  is  no  less  true  of  the  higher 
forms,  since  in  all  of  them  an  internal  medium,  or  environment,  of 
a  liquid  nature,  the  lymph,  the  blood  or  sap,  is  found  which  is  the 
immediate  environment  of  the  cells.  Water  is  the  largest  and  one 
of  the  most  important  constituents  of  living  matter,  and  if  organisms 
are  carefully  examined  the  most  various  devices  are  found  to  assure 
the  regulation  of  the  water  content  of  the  cells  of  the  body.  The 
younger,  the  more  vigorous,  the  more  alive,  the  more  actively 
growing,  the  more  impressible  cells  are,  the  more  watery  are  they." 

Water  enters  very  largely  into  the  composition  of  the  bacterial 
cell,  since  they  consist  of  from  70  to  95  per  cent,  water;  moreover, 
it  enters  into  nearly  every  change  which  they  bring  about.  When 
5 


66  FOOD  REQUIREMENTS 

bacteria  decompose  the  carbohydrates,  one  or  more  molecules  of 
water  are  taken  up;  when  they  synthesize,  water  is  eliminated. 
The  hydrolysis  of  fats  requires  for  every  molecule*  three  of  water; 
when  they  are  synthesized  from  glycerin  and  fatty  acids  three 
molecules  are  eliminated.  The  digestion  of  the  proteins  by  bacteria 
is  usually  hydrolysis  in  which  a  number  of  molecules  of  water  are 
caused  to  enter  the  large  protein  molecule,  thus  causing  it  to  break 
down  into  elementary  diffusible  foods.  When  the  bacteria  build 
their  own  proteins  from  the  peptones  and  amino-acids,  it  requires 
that  water  be  eliminated.  Thus  water  plays  an  all-important  part 
in  all  bacterial  syntheses  and  decompositions. 

Water  accelerates  or  is  essential  in  all  reactions  taking  place  in 
the  cell.  It  has  a  higher  inductive  capacity,  or  dielectric  constant, 
than  any  other  liquid,  except  possibly  hydrogen  dioxid.  "It  is  a 
good  insulator.  It  does  not  in  itself,  at  ordinary  temperatures, 
conduct  the  current  readily.  In  virtue  of  this  property  it  happens 
that  when  electrical  disturbances  occur  in  a  cell  they  are  not 
instantly  compensated,  so  that  oppositely  charged  particles  may 
coexist  in  water.  It  is  probably  because  of  this  property  that 
water  forms  such  a  good  ionizing  medium.  At  any  rate,  this 
property  may  account  for  the  undoubted  fact,  whatever  explanation 
we  may  choose  to  give  of  that  fact,  that  substances  dissolved  in 
water  interact  with  greater  ease  and  speed  than  when  dissolved  in 
any  other  medium.  It  has  the  property  then,  so  important  for  the 
cell,  of  accelerating  all  kinds  of  chemical  reaction.  Thus  hydro- 
gen and  oxygen  will  not  unite,  except  at  very  high  temperatures, 
unless  some  water  is  present.  Hydrochloric  acid  and  sodium 
hydrate  react  vigorously  in  the  presence  of  water,  but  not  when  they 
are  quite  dry.  Chlorin  and  hydrogen  do  not  form  hydrochloric 
acid,  except  at  very  high  temperatures,  unless  water  be  present,  and 
everyone  knows  that  the  rusting  of  iron  does  not  occur  unless  water 
is  there  too.  Water  has,  then,  this  fundamental  property  of 
making  reactions  go  on  between  bodies  dissolved  in  it  or  wet  by  it. 
This  property  is  believed  by  many  to  be  correlated  with  its  ionizing 
powers  and  with  the  fact  that  its  solutions  conduct  electrical  currents 
more  than  those  of  any  other  solvent." 

Another  very  remarkable  property  of  water  is  its  power  of  solu- 
tion. No  other  solvent  surpasses  it.  All  substances  dissolve  in  it 
to  some  extent.  It  is  a  solvent  for  salts,  carbohydrates,  proteins 
and  even  for  fats  to  some  extent.  This  universal  solvent  power 
has  not  yet  been  fully  explained,  but  it  is  probable  that  it  is  cor- 
related with,  or  due  to,  the  extra  valances  on  the  oxygen  atoms 
which  are  perhaps  able  to  unite  with  the  extra  valances  on  the  dis- 
solving molecules  and  thus  to  produce  solution.  But  be  the  expla- 
nation what  it  may,  it  is  well  known  that  its  solvent  action  con- 
tributes much  to  life.  Bacteria  are  able  to  absorb  their  food  only 


KIND  OF  FOOD  REQUIRED  67 

when  in  solution,  while  in  solution  it  reacts  and  after  it  has  served 
its  purpose  the  waste  products  are  carried  from  the  cell  in  solution. 

Osmotic  Pressure.— If  a  cell  be  placed  in  a  strong  salt  solution, 
there  is  a  shrinking  of  the  cell  which  may  result  in  plasmolysis. 
If,  on  the  other  hand,  a  cell  be  suspended  in  pure  water  the  cell 
greatly  increases  in  size  and  finally  bursts.  This  is  the  case  when 
any  solution  is  separated  from  pure  water  or  from  a  less-concen- 
trated solution  by  a  membrane  which  to  the  dissolved  substance  is 
impermeable  but  to  the  water  of  .the  solution  permeable.  The  solu- 
tion exerts  pressure  on  the  membrane  and  the  water  passes  through 
the  membrane  into  the  solution.  The  pressure  is  called  osmotic 
pressure,  and  depends  not  upon  the  percentage  of  solute,  but  upon 
the  number  of  particles— molecules  and  ions— per  unit  volume.  The 
amount  of  this  pressure  varies  in  different  cells,  but  for  mammals 
it  is  supposed  to  be  about  that  of  a  0.9  per  cent,  sodium  chlorid 
(NaCl)  solution,  since  in  such  a  solution  the  tissue  neither  gains  nor 
loses  weight.  This  is  about  7.1  atmospheres. 

However,  some  bacteria  and  many  molds  can  survive  and  even 
grow  in  salt  solution  which  would  be  fatal  to  the  life  of  the  cell  of 
higher  plants.  Penicillium  and  Aspergillus  have  been  known  to 
thrive  in  solutions,  the  osmotic  pressure  of  which  is  equivalent 
to  a  20  per  cent,  potassium  nitrate  solution.  Bacillus  anthracis 
flourishes  on  agar  containing  as  much  as  from  8  to  10  per  cent,  of 
sodium  chlorid.  Since  turgidity  is  essential  to  growth,  it  follows 
that  these  organisms  must  have  some  means  of  altering  the  pressure 
of  their  cell  contents  according  to  the  concentration  of  the  sur- 
rounding medium;  only  in  this  way  can  plasmolysis  be  avoided. 
The  plasmotic  membrane  in  the  case  of  many  bacteria  is  highly 
permeable;  this  would  be  the  case,  especially  with  those  organisms 
which  grow  in  brines.  Even  some  pathogenic  bacteria  possess  the 
power  of  accommodating  themselves  to  high  osmotic  pressures. 
Bacillus  cholera  are  temporarily  plasmolyzed  by  salt  and  sucrose 
solutions  but  not  at  all  by  a  glycerin  solution,  the  cell  membrane 
being  permeable  to  the  latter.  The  plasmolysis  produced  by  the 
salt  and  sugar  disappear  in  the  course  of  an  hour  or  two  as  a  rule, 
showing  that  even  salt  and  sugar  slowly  penetrate  the  plasmotic 
membrane. 

Kind  of  Food  Required. —The  quality  of  the  food  required  by 
bacteria  varies  greatly  with  the  species.  This  is  well  exemplified 
in  Jensen's  classification  of  bacteria  which  is  based  upon  the  sources 
of  nutrition  and  distinguishes  the  following  groups: 

"1.  Bacteria  which,  like  green  plants,  need  neither  organic 
carbon  nor  organic  nitrogen.  These  so-called  '  autotrophic  bacteria' 
can  build  up  both  carbohydrates  and  proteins  out  of  carbon  dioxid 
and  inorganic  salts. 

"2,  Bacteria  which  need  organic  carbon  compounds,  but  can 


68  FOOD  REQUIREMENTS 

dispense  with  organic  nitrogen.  These  bacteria  are  able  to  synthe- 
size protein  substances  out  of  carbohydrates  (or  organic  acids)  and 
ammonia,  nitrogen  or  nitrates. 

"3.  Bacteria  which,  like  the  higher  animals,  require  both  organic 
carbon  and  organic  nitrogen  compounds.  These  bacteria  cannot 
accomplish  either  carbohydrate  or  protein  synthesis  out  of  inorganic 
substances." 

Carbon.— The  carbon  dioxid  of  the  air  cannot  be  utilized  by 
bacteria  as  a  source  of  energy  since  it  is  already  fully  oxidized. 
There  are,  however,  some  organisms  which  possess  the  power  of 
utilizing  both  carbon  monoxid  and  methane.  On  the  contrary, 
the  carbon  of  carbohydrates,  fats  and  proteins  are  readily  utilized 
by  bacteria.  The  hydrocarbo~ns  of  both  the  aliphatic  and  aromatic 
series  are  resistant  to  bacteria,  but  those  compounds  which  contain 
oxygen  in  addition  to  the  carbon  and  hydrogen  are  more  readily 
attacked.  Many  organic  acids  and  oxy-acids  are  used  by  some 
bacteria.  Only  a  few  bacteria  can  use"  the  simpler  alcohols.  The 
more  complex  alcohols,  like  glycerin  and  mannite,  are  utilized 
by  many.  The  carbohydrates  are  especially  valuable  to  most 
bacteria,  those  containing  six  or  twelve  carbon  atoms  being  the 
most  valuable. 

Nitrogen. —The  nature  of  the  nitrogen  requirements  of  bacteria 
are  extremely  different,  depending  upon  the  specific  organism. 
Some  organisms,  such  as  the  symbiotic  nitrogen-fixers  and  the 
azofiers,  are  able  to  obtain  all  the  nitrogen  required  from  the 
atmosphere.  The  nitrosomonas  obtains  its  nitrogen  from  ammonia, 
whereas  the  nitromonas  obtain  it  from  nitrites.  The  majority  of 
bacteria  obtain  their  nitrogen  from  peptones,  proteoses.  and  even 
amino-acids.  Rettger  concludes  from  oft-repeated  experiments  on 
animal  and  vegetable  proteins  that  bacteria  are  unable  to  derive 
nourishment  from  native  proteins,  and  that  in  a  medium  in  which 
there  is  no  possible  source  of  nitrogen  other  than  the  proteins 
themselves  they  will  thrive  no  better  than  in  a  chemically  pure 
saline  solution.  When  proteolytic  enzymes  are  present  the  com- 
plex protein  molecules  are  broken  up  and,  at  least  in  part,  made 
available  for  cell  nutrition.  It  would  appear  that  "it  is  as  essen- 
tial to  break  down  complex  nitrogenous  food  substances  into  their 
simple  components,  before  they  can  be  utilized,  as  it  is  to  reduce  the 
walls  of  an  old  church  brick  by  brick  before  they  can  be  made 
over  into  a  modern  schoolhouse."  The  more  strictly  pathogenic 
organisms,  as  the  gonococcus  and  the  leprosy  bacillus,  may  require 
nitrogen  in  the  form  of  highly  specific  tissue  proteins.  As  a  rule, 
animal  proteins  are  more  readily  utilized  than  are  plant  proteins. 

Hydrogen.— Hydrogen  is  obtained  from  many  organic  compounds 
containing  hydrogen  and  oxygen,  such  as  the  carbohydrates,  fats 


OXYGEN  REQUIREMENTS  69 

and  ^proteins,  but  usually  not  those  compounds  which  contain  only  | 
carboirand  hydrogen,  such  as  methane  and  its  homologues. 

Sulphur.  —Sulphur  is  required  by  all  bacteria  possibly  for  the  for- 
mation of  the  proteinaceous  material  of  their  bodies.  In  addition 
to  this,  some  organisms  use  it  as  a  source  of  energy.  For  instance, 
the  Beggiatoa  sometimes  use  two  to  four  times  their  own  weight  of 
hydrogen  sulphid  in  a  day,  under  which  conditions  the  sulphur  grains 
may  be  seen  in  the  cell-protoplasm  and  may  be  looked  upon  as  an 
intermediate  stage  in  the  oxidation  process,  the  reaction  proceeding 
as  follows: 

2H2S     +      O2      =     2H2O     +     2S 
2S        +     3O2     +     2H2O      =     2H2SO4 

Some  bacteria  may  get  their  required  sulphur  from  sulphates,  sjjjr 
.phites  or  thiosulphates.  but  probably  the  great  majority  of  them 
obtain  it  from  the  proteins. 

Phosphorus.— Phosphorus  is  used  by  bacteria  in  large  quantities, 
being  essential  for  the  building  of  the  nucleoproteins  and  phospho- 
proteins  in  which  the  unicellular  organisms  are  especially  rich. 
The  form  and  quantities  required  by  the  organisms  vary  greatly 
with  the  species.  The  Azotobacter  are  able  to  utilize  it  from  most 
jorganic  and  inorganic  sources,  some,  however,  being  much  more 
valuable  than  others. 

Potassium.— Potassium  is  essential  to  the  higher  plants  and  cannot 
be  replaced  entirely  by  related  substances,  yet  Gerlach  and  Vogel 
early  reached  the  conclusion  that  potassium  and  magnesium  are 
not  essential  to  Azotobacter.  Their  results,  however,  were  con- 
sidered for  a  long  time  to  be  erroneous.  But  if  these  elements  are 
essential  to  Azotobacter  it  must  be  in  extremely  small  quantities. 
Potassium  does,  however,  favor  their  development  and  is  probably 
valuable,  if  not  essential,  to  all  bacteria.  Most  inorganic  potassium 
compounds  can  be  utilized. 

Other  Inorganic  Substances. —The  other  inorganic  constituents 
are  required  by  bacteria  only  in  small  quantities  and  are  obtained 
from  either  organic  or  inorganic  compounds,  depending  upon  the 
specific  organism. 

Oxygen  Requirements. —Bacteria,  like  all  other  plants  and  animals, 
require  oxygen  in  their  life  activity.  The  various  classes  of  organ- 
isms are  not  indifferent  as  to  the  form  in  which  they  obtain  their 
oxygen.  One  great  class  requires  that  their  oxygen  be  furnished 
free;  to  these  is  given  the  name  "aerobic."  Another  requires  their 
oxygen  in  the  combined  form;  they  are  called  "anaerobic."  Some 
organisms  grow  best  in  the  presence  of  free  oxygen  but  may  become 
adapted  to  combined  oxygen;  these  are  known  as  "facultative 
anaerobes."  Others  grow  best  in  the  absence  of  free  oxygen  but 
may  become  adapted  to  it;  they  are  known  as  "facultative  aerobes." 


70  FOOD  REQUIREMENTS 

Few  bacteria  are  true  aerobes  or  anaerobes,  but  many  gradually 
blend  from  one  class  into  another,  as  some  will  withstand  small 
quantities  of  free  oxygen  but  not  a  full  atmospheric  pressure  of  it. 

Vitamines.— The  extracts  of  animal  organs,  as  well  as  those  of 
some  plant  tissues,  are  valuable  nutrient  material  for  bacteria  which 
it  is  as  yet  impossible  to  supply  in  any  medium  of  known  chemical 
composition.  The  composition  of  these  more  or  less  unstable 
but  highly  nutritive  substances  is  a  matter  of  purest  speculation. 
For  want  of  a  better  name  they  are  termed  "vitamines"  or  "acces- 
sory growth  factors."  These  accessory  bodies  are  moderately 
heat-stable  and  are  soluble  in  alcohol  and  in  water.  They  are 
rapidly  absorbed  from  solution  by  filter  paper,  but  not  by  glass 
wool.  They  increase  the  reaction  velocity  of  the  proteolytic  metab- 
olism of  the  meningococcus  and  are  essential  to  many  other  organ- 
isms. After  the  first  or  primary  cultivation  some  organisms  become 
independent  of  these  substances.  This  phase  of  bacterial  nutri- 
tion, which  is  only  just  beginning  to  receive  attention,  is  beset  by 
many  difficulties.  The  work  being  done,  however,  gives  promise 
of  so  clearing  up  the  field  that  much  that  was  impossible  of  expla- 
nation in  the  past  will  be  readily  explained.  But  the  present 
status  of  the  case  is  well  summarized  by  Rettger  when  he  stated: 
"We  are  as  yet  in  the  dark  regarding  the  real  food  requirements 
of  bacteria." 

REFERENCES. 

Marshall:     Microbiology. 

Kendall:     Bacteriology — General,  Pathological  and  Intestinal. 

Kruse:     Allgemeine  Microbiologie. 

Herman,  Nathan,  and  Rettger,  Leo  F.:  Bacterial  Nutrition — Further  Studies 
on  the  Utilization  of  Protein  and  Non-protein,  Jour.  Bacteriol.,  1918,  iii,  367-388. 

Berman,  (Nathan),  and  Rettger  (Leo  F.):  The  Influence  of  Carbohydrates  on 
the  Nitrogen  Metabolism  of  Bacteria,  Jour.  Bacteriol.,  1918,  iii,  389-402. 


CHAPTER  VII. 
BACTERIAL  METABOLISM-ENZYMES. 

IT  was  pointed  out  in  the  last  chapter  that  bacteria  require  food 
for  at  least  two  purposes— building  material  and  the  liberation 
of  energy.  In  fulfilling  these  functions  the  foods  are  profoundly 
changed;  at  times  they  are  broken  up  into  comparatively  simple 
products,  after  which  they  are  built  into  the  complex  molecules 
composing  the  bacterial  cell;  at  other  times  they  are  split  and  the 
energy  utilized;  at  still  other  times  they  are  completely  oxidized, 
the  organisms  thus  obtaining  all  the  stored  potential  energy.  The 
sum  of  all  these  changes  which  the  food  undergoes,  including  the 
deterioration  of  the  cell,  is  called  metabolism.  These  changes  con- 
sist of  two  separate  processes;  the  one — construction  of  new  cells 
or  parts  of  cells — is  a  process  of  synthesis  and  is  called  anabolism. 
The  other  is  analytical  or  the  breaking-down  of  the  cell  and  is 
called  katabolism.  Although  these  two  processes  are  usually  going 
on  simultaneously  in  the  cell,  yet  it  is  true  that  during  the  first  few 
hours  after  inoculation  of  a  culture  the  anabolic  aspect  predominates; 
later  the  katabolic  phase  predominates.  That  this  should  be  the 
case  can  be  readily  seen,  for  the  bacterial  cell  must  be  morphologi- 
cally complete  before  it  can  bring  about  its  characteristic  energy 
transformations,  which  continues  until  the  death  of  the  cell. 

Moreover,  recent  investigations  have  demonstrated  that  it  is 
just  as  true  of  bacteria  as  of  animals  that  "it  is  as  essential  to 
break  down  complex  nitrogenous  food  substances  into  their  simple 
components  before  they  can  be  utilized,  as  it  is  to  reduce  the  walls 
of  an  old  church  brick  by  brick  before  they  can  be  made  over  into 
a  modern  schoolhouse."  The  development  and  present  status  of 
our  knowledge  of  this  represents  one  of  the  most  interesting  and 
valuable  chapters  of  bacteriology. 

Early  Theories  of  Fermentation.— Even  as  early  as  1595  the  great 
medical  chemist,  Labavius,  considered  fermentation  a  process  akin 
to  digestion,  and  von  Helmont  (1648)  stated  that  out  of  the  ferment 
something  passes  into  the  fermenting  liquid  which  grows  in  it  as 
a  seed.  But  it  was  the  great  chemist,  Liebig,  who  first  developed 
the  purely  chemical  explanation  of  fermentation.  It  was  he  who 
developed  the  idea  of  catalysis,  a  word  already  invented  by 
Berzelius.  Liebig  compared  fermentation  changes  to  the  action 
of  finely  divided  platinum  which  possesses  the  power  of  bringing 
about  the  union  of  gases  at  low  temperatures.  The  ferment  he 


72  BACTERIAL  METABOLISM— ENZYMES 

considered  to  be  in  a  state  of  unstable  equilibrium*  or  decomposi- 
tion. This  is  communicated  to  its  surroundings,  producing  chemical 
changes.  This  was  opposed  by  Pasteur  and  Tyndall  who. showed 
that  in  the  absence  of  microorganisms  fermentation  does  not  take 
place. 

There  were  certain  changes  which  they  proved  to  be  due  to 
bacteria  and  yeast;  others  which  were  brought  about  by  pepsin, 
tripsin,  etc.  This  led  to  the  classification  of  ferments  as  organized 
and  unorganized.  Under  organized  ferments  were  grouped  such 
substances  as  some  bacteria  and  yeasts,  which,  when  examined 
under  the  microscope,  possess  a  definite  organized  structure  and 
which  act  by  virtue  of  vital  processes.  The  unorganized  ferments 
included  amylase,  pepsin,  rennin,  etc.,  and  were  described  as 
"non-living  unorganized  substances  of  a  chemical  nature."  Kiihne 
designated  this  last  class  of  substances,  enzymes.  This  classifica- 
tion into  organized  and  unorganized  ferments  was  generally  accepted 
and  practically  unquestioned  until  overthrown  by  Biichner  (1897) 
in  his  epoch-making  investigation  of  yeast.  He  carefully  mixed 
1000  grams  of  brewers'  yeast  with  an  equal  weight  of  quartz  sand 
and  250  grams  of  infusorial  earth  generally  known  as  Kieselguhr. 
This  mixture  was  ground  together  until  plastic;  100  c.c.  of  water 
was  added  and  wrapped  in  a  press  cloth  and  filtered  in  a  press  cap- 
able of  exerting  a  pressure  of  from  400  to  500  atmospheres.  The 
juice  was  clarified  by  shaking  with  Kieselguhr  and  filtering.  The 
liquid  so  obtained  is  slightly  heavier  than  water  and  possesses  a 
pleasant  odor.  On  boiling,  a  quantity  of  proteinaceous  matter 
separates  and  the  liquid  becomes  nearly  solid. 

The  unboiled  juice  possesses  all  the  power  of  the  yeast  cell  in  so 
far  as  fermentation  is  concerned.  However,  the  action  is  not 
stopped  by  chloroform  nor  by  the  passage  of  the  liquid  through  a 
Berkefeld  filter  nor  through  a  dialyzing  membrane.  The  enzyme 
which  is  present  in  the  solution  has  been  termed  by  Biichner 
zymase.  Later  the  lactic  acid-  and  the  acetic  acid-producing  bacteria 
were  subjected  by  Biichner  to  similar  treatment  to  that  given  the 
yeast  cells,  and  the  active  intracellular  enzymes  were  obtained. 
Since  that  time  the  list  of  unorganized  ferments  or  enzymes  has 
continued  to  grow  at  the  expense  of  the  organized  ferments  until 
it  is  generally  conceded  today  that  all  fermentations  are  due  to 
enzymes,  there  being  only  this  difference— that  some  are  formed 
and  readily  diffuse  out  of  the  body  of  the  eel)  during  its  life  and  are 
known  as  extracellular  ferments,  whereas  others  remain  in  the  cell 
and  are  known  as  intracellular  ferments. 

Definition  of  Enzymes.— Enzymes  have  been  defined  as  "unor- 
ganized, soluble  ferments,  which  are  elaborated  by  an  animal  or 
vegetable  cell  and  whose  activity  is  entirely  independent  of  any 
of  the  life  processes  of  such  a  cell." 


DEFINITION  OF  ENZYMES  73 

Enzymes  act  by  catalysis  and  hence  are  often  stated  to  be  "  select- 
ive colloidal  catalysts,  present  in  living  cells  and  destroyed  by 
heat."  A  catalyzer  is  "a  substance  which  alters  the  velocity  of 
a  chemical  reaction  without  undergoing  any  apparent  physical  or 
chemical  change  itself  and  without  becoming  a  part  of  the  product 
formed."  It  is  a  well-known  fact  that  the  speed  of  many  chemical 
reactions  is  accelerated  by  catalyzers;  for  example,  the  inversion  of 
cane  sugar  by  acid  and  the  numerous  reactions  affected  by  platinum. 
Negative  catalysis  is  not  as  common,  but  the  stopping  of  theslow 
oxidation  of  phosphorus  in  air  by  a  trace  of  ether  vapor  may  be 
taken  as  an  example.  The  general  characteristics  of  catalysts  are 
admirably  illustrated  by  Bayliss: 

"There  are  certain  phenomena  which,  at  first  sight,  might  be 
confused  with  those  of  catalysis,  but  which  must  be  carefully  dis- 
tinguished from  them.  A  mechanical  model  will  serve  to  make 
this  clear.  If  a  brass  weight  of,  say  500  grams,  be  placed  at  the 
top  of  an  inclined  plane  of  polished  plate-glass,  it  will  be  possible 
to  find  a  slope  of  the  plane  such  that  the  weight  will  slowly  slide 
down.  This  represents  any  reaction  taking  time  to  complete. 
If  now  the  bottom  of  the  weight  be  oiled  (oil-catalyst)  the  rate  of 
its  fall  will  be  greatly  increased.  We  see,  that  in  either  case,  the 
weight  if  placed  at  the  top  of  the  plane  does  not  remain  there,  but 
sooner  or  later  reaches  the  bottom.  It  may,  however,  be  kept  at 
the  top  by  some  kind  of  catch  or  trigger  arrangement,  in  which  case 
it  will  remain  there  indefinitely  until  the  catch  is  released.  The 
amount  of  energy  lost  by  the  weight  in  its  fall,  being  the  product 
of  its  weight  and  the  vertical  height  from  which  it  has  fallen,  is  in 
no  way  affected  by  the  work  required  to  remove  the  obstacle  pre- 
venting its  fall,  nor  is  the  rate  at  which  it  falls  when  set  free.  A 
typical  instance  of  such  a  'trigger'  action  is  that  of  supersaturated 
solutions,  which  remain  for  any  length  of  time  unchanged  unless 
infected  with  a  crystal.  It  has,  moreover,  been  shown  by  B. 
Moore  (1893)  that  the  rate  at  which  the  solidification  of  supercooled 
glacial  acetic  acid  moves  along  a  tube  is  independent  of  the  quantity 
of  crystals  placed  at  one  end  to  start  the  process.  Not  so  with 
true  catalytic  action;  although  the  work  done  by  our  sliding  weight 
is  in  no  way  affected  by  the  amount  of  catalyst  (oil)  used,  the  rate 
of  the  fall  is,  within  limits,  directly  proportional  to  it,  and  this  is  a 
property  of  catalysts  in  general. 

"It  cannot  be  expected  that  a  rough  model  of  this  kind  would 
show  all  of  the  characteristics  of  catalytic  phenomena,  but  there 
are  two  instructive  points  shown  by  it  in  addition  to  those  already 
spoken  of.  The  first  is  the  disappearance  of  the  catalyst  by  stick- 
ing to  the  glass  as  the  weight  slides  down.  An  analogous  phe- 
nomenon is  often  met  with  in  catalytic  processes,  as  will  be  seen 
later.  The  second  point  is  one  of  importance  with  regard  to 


74  BACTERIAL  METABOLISM— ENZYMES 

certain  enzyme  actions;  it  consists  in  the  fact  that,  although  the 
presence  of  the  catalyst  neither  adds  to  nor  subtracts  from  the 
total  energy  change  in  the  reaction,  the  form  of  this  energy  may  be 
altered.  When  the  weight  falls  slowly  by  itself,  nearly  the  whole 
of  the  energy  appears  as  heat  due  to  friction  along  the  glass  plane, 
so  that  the  weight  arrives  at  the  bottom  with  very  little  kinetic 
energy;  on  the  contrary,  when  oiled,  nearly  the  whole  of  the  energy 
is  present  in  the  weight  at  the  end  of  its  fall  as  kinetic  energy,  very 
little  friction  having  been  met  with  in  its  descent.  We  may  notice, 
also,  comparing  the  effects  of  different  amounts  of  oil,  that  small 
amounts  produce  a  much  more  marked  result  than  the  subsequent 
addition  of  further  quantities.  This  is  also  characteristic  of 
enzymes,  as  we  shall  see  later. 

"From  what  has  been  said  it  follows  that  a  catalyst  is  merely 
capable  of  changing  the  rate  of  a  reaction  already  in  progress.  In 
opposition  to  this  it  may  reasonably  be  said  that  a  reaction  does 
sometimes  seem  to  be  initiated.  Such  a  case  is  that  of  a  mixture 
of  oxygen  and  hydrogen  gases  caused  to  combine  by  spongy  plati- 
num. Now  there  are  reasons  for  the  belief  that  an  extremely  slow 
combination  is  taking  place  at  ordinary  temperatures  without 
catalysis.  One  thing  to  be  considered  in  reference  to  this  belief 
is  the  enormous  acceleration  of  chemical  reactions  by  rise  of  tem- 
perature, the  majority  being  about  doubled  by  a  rise  of  10°  C.  In 
this  way  a  reaction  having  a  velocity  of  1  at  0°  would  reach  one 
of  2  at  10°,  4  at  20°  and  1  X  210  =  1024  at  100°.  At  the  tempera- 
ture of  500°  there  is  appreciable  formation  of  water  in  the  case  in 
point,  and  Bodenstein  (1899)  has  shown  that  if  the  velocity  at  689° 
be  represented  by  163,  that  at  482°  has  already  sunk  to  0.28;  so  that 
at  room  temperature  the  velocity  would  be  quite  incapable  of 
detection  by  chemical  means,  since  centuries  would  be  needed 
to  produce  a  fraction  of  a  milligram  of  water.  Grove's  gas  battery 
also  proves  that  the  two  gases  are  not  in  equilibrium  at  ordinary 
temperatures,  since  electrical  energy  is  obtained  by  their  slow  com- 
bination. 

"To  take  another  case  of  a  reaction  which  progresses  at  a  slow 
rate  when  left  to  itself:  When  methyl  acetate  is  mixed  with  water 
at  ordinary  temperatures  it  is  very  slowly  hydrolyzed  to  alcohol 
and  acetic  acid  until  a  certain  proportion  of  it  is  decomposed,  so 
that  a  state  of  equilibrium  is  finally  arrived  at.  This  process  takes 
many  days  for  its  completion,  but  the  time  may  be  shortened  to  a 
few  hours  by  the  addition  of  a  small  amount  of  hydrochloric  acid. 

"The  objection  may  be  made  to  the  former  of  these  two  examples 
that  the  combination  of  oxygen  and  hydrogen  does  not  take  place 
except  in  the  presence  of  water  vapor,  which  probably  acts  as  a 
catalyst.  Similarly,  the  hydrolysis  of  esters  by  water  may  be  said 
to  be  due  to  the  hydrion  present  therein.  This  point  of  view  does 


TERMINOLOGY  75 

not,  however,  in  reality,  affect  the  reasoning,  since  the  reactions 
can  be  enormously  accelerated  by  other  bodies,  which  act  as  addi- 
tional catalysts  and  may  be  investigated  independently.  It  is,  in 
fact,  a  matter  of  considerable  difficulty  to  discover  a  slow  reaction 
which  is  definitely  known  to  take  place  in  the  complete  absence  of 
any  catalyst. 

"Moreover,  it  must  not  be  forgotten  that,  as  J.  J.  Thomson  and 
others  believe,  a  catalyst  may  possibly  start  a  reaction.  This  is 
not,  theoretically,  in  disagreement  with  the  view  taken  by 
Ostwald.  To  return  to  our  mechanical  illustration,  the  ' friction' 
between  the  weight  and  the  glass  plane  may  be  sufficiently  great 
to  prevent  movement  altogether,  until  oil  is  applied.  But  the  use 
of  the  name  'friction'  implies  the  idea  of  movement  and  the  exist- 
ence of  forces  tending  to  produce  it.  One  may  indeed  suppose 
that  the  weight  actually  does  move  for  an  infinitesimal  distance, 
but  is  at  once  arrested  by  the  resistance  met  with.  From  thi? 
point  of  view  the  definition  of  a  catalyst  would  be  expressed  some- 
what thus :  A  substance  which  ch  anges  the  rate  of  a  reaction  which 
is  actually  in  progress,  or  which  is  capable  of  proceeding  without 
any  supply  of  energy  from  without,  if  certain  resisting  influences 
are  removed.  The  difference  between  diminution  of  friction  by 
oil  and  the  removal  of  a  catch  is  that,  in  the  former  case  the  action  is 
continuous  throughout  the  fall  of  the  weight,  whereas  in  the  latter 
case  the  action  is  only  momentary,  at  the  commencement  of  the 
fall,  on  the  rate  of  which  it  has  no  further  effect." 

Terminology.— Within  recent  years  attempts  have  been  made  to 
systematize  the  terminology  used  in  referring  to  enzyme  action. 
The  name  of  the  substance  on  which  the  enzyme  acts  is  called 
substrate. 

As  to  the  names  of  the  enzymes  themselves  it  is  customary  to 
use  the  termination  "  ase"  which  denotes  an  enzyme  and  this 
termination  should  be  added  to  the  root  of  the  word  which  names 
the  substrate;  for  example,  lactase  is  the  enzyme  accelerating  the 
hydrolysis  of  lactose,  sucrase  of  sucrose,  maltase  of  maltose,  etc. 
Unfortunately,  in  many  cases  old  names  have  become  so  fixed  that 
it  is  not  desirable  to  replace  them,  as,  for  example,  pepsin  for  the 
acid  proteinase  and  trypsin  for  the  alkali  proteinase.  At  other 
times  the  enzymes  are  incorrectly  named  from  the  simpler  substance 
in  place  of  the  more  complex  substrate;  for  example,  invertase  for 
the  ferment  which  inverts  sucrose. 

It  is  the  custom  with  many  writers  to  speak  of  the  enzymes  which 
attack,  say,  starch  or  protein,  as  "amylolytic"  or  "proteolytic," 
respectively;  but  Armstrong  has  pointed  out  that  these  names 
are  incorrectly  formed.  "Amylolytic"  in  analogy  with  "electrolytic" 
should  mean  a  decomposition  by  means  of  starch.  To  avoid  this 
misuse  of  words  he  advocates  the  use  of  the  termination  "  clastic" 


76  BACTERIAL  METABOLISM— ENZYMES 

instead  of  "lytic,"  giving  us  terms  such  as  "amyloclastic,"  "proteo- 
clastic,"  "lipoclastic,"  etc. 

Enzymes  ordinarily  do  not  occur  active  within  the  cell,  but 
are  present  in  the  form  of  a  zymogen  or  mother  substance.  This 
substance,  when  acted  upon  by  a  specific  substance,  becomes 
active  and  the  process  is  termed  "activation."  The  agency  which 
is  instrumental  in  activating  a  zymogen  is  termed  "  zymo-excitor" 
or  kinase. 

Properties  of  Enzymes.— Enzymes  are  known  from  the  reactions 
which  they  catalyze  and  they  are  found  to  follow  quite  definite 
laws  in  their  reactions.  Some  of  the  more  important  are  as  follows: 

1.  An  enzyme  does  not  initiate  a  chemical  reaction  but  only 
alters  its  velocity;  nor  does  it  appear  in  the  final  products  of  the 
reaction  which  it  accelerates.  We  must,  therefore,  assume  that 
substances  are  slowly  changing  and  that  the  catalyst  does  nothing 
more  than  alter  the  speed  of  this  reaction.  The  state  of  affairs  is, 
therefore,  similar  to  that  of  a  mixture  of  oxygen  and  hydrogen 
gases  catalyzed  by  platinum  in  which  there  is  evidence  that  the 
combination  takes  place  at  room  temperatures,  although  at  an 
unmeasurable  rate.  Salicin,  which  is  readily  hydrolized  by  ptyalin 
and  emulsin  to  glucose  and  saligenin  slowly  decomposes  in  water 
at  150°  C.  It  would,  therefore,  be  inferred  that  the  process  also 
takes  place  at  room  temperature.  Starch  solutions  slowly  undergo 
a  spontaneous  change  into  dextrin  and  sugar  and  solutions  of 
ammonium  caseinogenate  increase  in  electrical  conductivity  when 
left  to  themselves,  a  change  similar  to  that  which  occurs  when 
they  are  acted  upon  by  trypsin.  Taylor  has  shown  that  an  appreci- 
able proportion  of  pure  sterile  globulin  kept  in  distilled  water  at 
ordinary  temperature  for  eighteen  months  is  hydrolyzed  to  protease 
and  that  leucin  may  be  recovered  from  a  sterile  suspension  of  casein 
in  pure  water  and  that  arginin  may  be  recovered  from  a  solution 
of  protamin  sulphate  in  pure  water.  True,  the  reaction  is  slow  and 
the  products  have  accumulated  only  in  small  quantities  after  the 
lapse  of  a  year;  nevertheless,  it  is  evident  that  the  process  is  slowly 
occurring  in  the  absence  of  the  catalyzer. 

It  is  likely  that  the  ferment  enters  temporarily  into  chemical 
combination  with  the  substance  acted  upon.  This  assumption  is 
made  on  the  ground  that  the  sensitiveness  of  the  enzyme  often 
changes  when  brought  in  contact  with  the  substrate  and  may  at 
first  be  hard  to  separate.  Moreover,  it  is  definitely  known  that  in 
some  simple  catalytic  processes  the  catalyzer  does  temporarily 
combine  with  the  reacting  substance.  This  is  the  case  in  the 
manufacture  of  sulphuric  acid,  where  steam,  sulphur  dioxid,  oxygen 
and  the  oxids  of  nitrogen  are  introduced  simultaneously  into  a 
large  chamber  when  the  following  reactions  probably  occur. 

502  +  N2O3    =     SO3   +  2NO 

503  +  H20     =     H2SO4 
2NO     +  O»        =  2NO2 


PROPERTIES  OF  ENZYMES  77 

Thus  it  is  that  the  oxids  of  nitrogen  serve  to  convert  the  sulphur 
dioxid  to  the  trioxid  and  in  the  presence  of  air  reverts  to  the  original 
condition  and  again  repeats  the  cycle.  While  in  the  Gay-Lussac 
tower  the  nitrosul-sulphuric  acid  is  formed : 

N2O«+  H2SO4  =  2NO2HOSO2+  H2O 
2NO2HOSO2+  2H2O  =  2H2SO4+  N2O3 

Where  there  are  a  number  of  steps  in  a  reaction,  as  is  the  case  with 
the  above,  it  is  necessary,  as  pointed  out  by  Ostwald,  that  the  sum 
of  all  the  reactions  in  the  catalyzed  system  are  more  rapid  than  are 
the  changes  in  the  uncatalyzed. 

The  classic  illustration  of  an  organic  reaction  of  this  type  is  that 
afforded  by  the  production  of  ether  from  alcohol.  In  this  process 
sulphuric  acid  is  employed  as  catalyzer  and  as  well  known  this  first 
combines  with  alcohol  with  the  formation  of  ethyl-sulphuric  acid. 

HO  C2H5O 

\  \ 

C2H6OH     +  SO2     =     HOH     +  SO2 

/  / 

HO  HO 

Alcohol.  Sulphuric  acid.  Ethyl-sulphuric  acid. 

The  ethyl-sulphuric  acid  reacts  with  another  molecule  of  alcohol 
forming  ether  and  regenerating  sulphuric  acid. 

C2H6O  HO 

\  \ 

SO2     +     C2H5OH  =  SO2     +     C2H5  O     -     C2H5 

/  / 

HO  HO 

Ethyl-sulphuric  acid.     Alcohol.  Sulphuric  acid.  Ether. 

Similar  combinations  occur  with  the  enzymes,  for  it  is  found  that 
sucrase  will  withstand  uninjured  a  temperature  25°  C.  higher  in 
the  presence  of  sucrose  than  in  its  absence.  It  is  difficult  to  see 
how  this  could  happen  unless  the  enzyme  entered  into  some  sort  of 
union  with  the  sugar. 

Intimately  connected  with  the  subject  of  combination  of  enzyme 
with  substrate  is  that  of  specificity,  an  example  of  which  is  seen 
in  the  fact  that  certain  enzymes  act  only  on  carbohydrates,  others 
on  fats,  and  still  others  on  proteins.  The  group  of  those  trans- 
forming carbohydrates  is  further  subdivided  into  specific  enzymes 
each  of  which  has  the  power  of  acting  alone  upon  only  one  sugar. 
This  property  is  so  specific  that  in  many  cases  the  enzyme  will 
act  upon  one  optically  active  compound  leaving  the  opposite  optical 
isomer  untouched.  This  led  Fischer  to  the  formulation  of  his 
famous  simile  of  the  "lock  and  key"  relationship.  In  this  he 
considers  that  the  enzyme  and  its  substrate  must  have  an  inter- 
relation, such  as  the  key  has  to  the  lock;  otherwise,  the  reaction 


78  BACTERIAL  METABOLISM— ENZYMES 

does  not  occur.  By  means  of  this  theorem  it  has  been  possible  to 
foretell  the  structure  of  many  complex  substances  and  explain 
hitherto  obscure  points  in  biology. 

2.  The  chemical  change  brought  about  by  an  enzyme  in  infinite 
time  is  independent  of  the  concentration  of  the  enzyme,  but  for 
shorter  periods  it  is  clearly  and  usually  a  definite  function  of  the 
concentration  of  the  enzyme.    This  means  that  a  small  quantity 
of  enzyme  will  bring  about  as  much  change  as  a  large  one,  provid- 
ing unlimited  time  is  given.    In  this  regard,  then,  enzyme  reac- 
tions differ  from  ordinary  reactions  in  that  they  do  not  follow  the 
law  of  mass  action.    This  may  be  illustrated  by  the  carrying  of 
brick  to  the  top  of  a  building  by  men.    Give  one  man  sufficient 
time  and  he  would  be  as  able  to  transfer  the  whole  pile  to  the  top 
as  would  a  group  of  men,  but  in  the  latter  case  the  time  occupied 
would  be  inversely  proportional  to  the  number  of  men  working. 
So  it  is  with  enzymes;  the  intensity  is  almost  directly  proportional 
to  the  concentration  of  the  enzymes.     In  certain  instances  where 
this  generalization  has  been  found  not  to  hold,  attempts  have 
been  made  to  apply  the  Schutz-Borissow  Law— that  the  intensity 
of  enzyme  reaction  is  directly  proportional  to  the  square  root  of 
the  concentration.     But  even  this  generalization  does  not  hold,  for 
there  are  a  number  of  factors  which  tend  to  retard  or  accelerate 
enzyme  action.     Chief  among  these  which  retard  are  (a)  reversi- 
bility,   (6)    combination   of  enzyme  with  products,    (c)    negative 
autocatalysis,  which  with  the  previous  factor  leads  to  reversible 
inactivation  of  the  enzyme,  (d)  destruction  or  similar  drastic  changes 
in  the  properties  of  the  enzyme.    Those  which  accelerate  are  as 
follows :    (a)  combination  of  the  whole  of  the  enzyme  with  the  sub- 
strate when  the  latter  is  in  relatively  large  excess,  (6)  positive 
autocatalysis. 

3.  Reactions  which  are  catalyzed  by  enzymes  are  reversible. 
It  has  been  conclusively  shown  in  the  case,  of  many  reactions  and 
is  generalized  for  others  that  where  a  reaction  is  being  catalyzed  by 
enzymes  it  is,  unless  the  products  so  formed  are  removed  from  the 
reaction  medium,  reversible.    This  is  illustrated  by  the  saponifica- 
tion  of  ethyl-butyrate  by  means  of  lipase. 

C3H7COOC2H5     +     H2O      =     C3H7COOH     +     C2H5OH 
Ethyl-butyrate.  Butyric  acid.  Ethyl  alcohol. 

Starting  with  a  definite  quantity  of  ethyl-butyrate,  after  a  time 
we  find  in  the  reacting  media  ethyl-butyrate,  butyric  acid  and 
ethyl  alcohol;  commencing  with  butyric  acid  and  ethyl  alcohol,  we 
obtain  the  same  products  as  in  the  first  case.  This  implies  that  the 
synthetic  reactions  which  are  going  on  in  the  cell  are  catalyzed  by 
the  same  enzymes  as  are  the  analytic  reactions;  hence  reactions 
that  are  catalyzed  by  enzymes  are  never  complete  unless  the  result- 
ing products  are  removed  as  fast  as  formed. 


HYDROLYTIC  ENZYMES  79 

4.  Enzymes  are  usually  characterized  by  great  sensitiveness  to 
comparatively  low  temperatures  and  to  many  poisons.  This  prop- 
erty formerly  was  used  to  determine  whether  or  not  a  reaction  was 
being  catalyzed  by  an  enzyme;  but  there  are  known  a  few  cases  in 
which  the  enzyme  is  not  destroyed  by  boiling  water.  The  great 
majority  of  all  enzymes  are,  however,  destroyed  by  a  temperature 
somewhat  below  100°  C.,  many  even  as  low  as  60°  C.  This  property 
is  no  doubt  due  to  the  colloidal  nature  of  the  ferment  which,  on 
being  heated,  coagulates—  probably  much  as  does  a  protein,  for  it 
is  well  known  that  enzymes  are  more  sensitive  in  the  presence  of 
water  than  in  its  absence. 

Although  the  addition  of  hydrocyanic  acid  or  formaldehyde  to 
a  media  in  which  reactions  are  being  catalyzed  by  enzymes  puts 
a  stop  to  the  reaction,  yet  the  concentration  necessary  is  usually 
greater  than  that  which  can  be  borne  by  the  living  protoplasm. 
This  makes  it  possible  to  kill  the  cell  and  still  have  the  enzyme 
reactions  going  on  in  the  medium  by  carefully  adjusting  the  con- 
centration of  the  antiseptic  used. 

Various  methods  are  used  in  the  extraction  of  enzymes.  Some 
readily  diffuse  out  of  the  cell  and  may  be  taken  up  with  water; 
others  are  extracted  with  glycerin  or  acids;  in  still  other  cases  it 
is  necessary  to  decompose  completely  the  cells  as  did  Biichner 
in  obtaining  zymase.  The  resulting  product  is  then  often  purified 
by  alcoholic  or  other  precipitants.  This  drastic  treatment,  how- 
ever, often  impairs  the  activity  of  the  ferment. 

Classification.—  Fuhrmann  has  classified  enzymes  of  bacterial 
origin  into  four  types  as  follows: 

A.  Schizases  (hydrolytic)  cleavage  enzymes: 

1.  Carbohydrate-splitting  enzymes. 

2.  Glucoside-splitting  enzymes  (synaptase). 

3.  Fat-splitting  enzymes.    Lipases  (esterases). 

4.  Proteases,  protein-splitting  enzymes,  pepsin,  trypsin 

(lysins,  coagulases). 

B.  Fermentation  enzymes: 

Zymase  urease,  lactic-acid  enzyme. 

C.  Oxidizing  enzymes: 

Tyrosinase,  acetic  bacteria,  oxidase. 

D.  Reducing  enzymes: 

Reductase. 

Hydrolytic  Enzymes.—  As  a  type  of  the  hydrolytic  enzymes  which 
act  upon  carbohydrates,  we  may  take  maltase  which  converts 
maltose  into  dextrose  according  to  the  following  equation: 


Ci2H22On     +     H2O      =     CeHnOe     + 
Maltose.  Dextrose.  Dextrose. 

Maltase  is  an  enzyme  which  occurs  in  yeast,  many  bacteria,  and 
numerous  other  cells.     It  is  of  special  interest  inasmuch  as  it  is 


80  BACTERIAL  METABOLISM—  ENZYMES 

the  first  case  of  reversible  action  that  was  studied.  Craft  Hill 
found  that  the  addition  of  maltase  to  a  very  concentrated  solution 
of  dextrose  resulted  in  the  formation  of  a  disaccharid.  This  he  at 
first  thought  was  a  simple  reversion  of  dextrose  into  maltose,  but 
later  work  showed  that  the  sugar  formed  was  an  isomer  of  maltose. 
The  essential  fact,  however,  remained  that  the  one  enzyme  possessed 
both  synthetic  and  analytic  properties. 

Emulsion  is  an  enzyme  which  possesses  the  power  of  decompos- 
ing mandelic-nitrile-glucose  into  glucose,  benzaldehyde,  and  hydro- 
cyanic acid.  The  mandelic-nitrile-glucose  is  obtained  by  the  action 
of  maltase  upon  the  glucoside  amygdalin.  The  total  change 
brought  about  by  the  two  ferments  is  indicated  by  the  following 
equation: 


C2oH27NOn     +     2H20      =     C6H8CHO     +     HCN     + 
Amygdalin.  Benzaldehyde.     Hydrogen  cyanid.     Glucose. 

Lipases  act  upon  the  neutral  fats  and  are  widely  distributed  in 
both  plant  and  animal  cells.  They  bring  about  a  reaction  which 
may  be  expressed  by  the  following  general  reaction,  where  R  = 
the  residue  of  a  fatty  acid. 

GHz—  R  CH2OH 

I  I 

CH—  R     +     3H2O      =     3RH     +     CHOH 

I  I 

CH*—  R  CH2OH 

One  molecule  of  neutral  fat  is  split  into  three  molecules  of  fatty 
acid  and  one  of  glycerin.  This  is  the  general  reaction  which  occurs 
in  the  spoiling  of  butter  or  fat  due  to  bacterial  activity. 

Proteases,  which  possess  the  power  of  splitting  proteins,  are 
widely  distributed  in  bacteria,  as  is  exemplified  by  their  gelatin- 
liquefying  powers.  This  also  is  a  hydrolytic  reaction  in  which  a 
number  of  molecules  of  water  is  caused  to  enter  the  protein  molecule 
with  its  subsequent  breaking  down  into  proteoses,  peptones,  and 
finally  amino-acid.  Even  this,  as  complex  a  reaction  as  it  is,  has 
been  shown  to  be  reversible  in  at  least  two  cases. 

Zymases,  which  occur  in  the  yeast  cell,  are  endo-enzymes  and 
their  function  is  to  liberate  energy  for  the  use  of  the  cell,  as  is  shown 
by  the  following  table  from  the  work  of  Rahn  : 

ENERGY  LIBERATED   FROM    1    GRAM   OF  SUBSTANCE. 

Soluble  enzymes.  Endozymes. 

Pepsin,  trypsin       ...      0  calories         Lactacidase     ....      80  calories 

Lipase    ......      4        "  Alcoholase       .      .      .      .120 

Maltase  sucrase     ...    10        "  Urease        .....    230        " 

Lactase        .....    23        "  Vinegar  oxidase    .      .      .  2500        " 

The  first  zymase  isolated  from  a  microorganism  was  that  of 
urease,  or  the  ferment  which  converts  urea  into  ammonium  car- 


OXIDIZING  ENZYMES  81 

bonate,  and  which  was  shown  by  Musculus  to  be  present  in  the  dead 
cells  of  Micrococcus  urece  which  develops  in  putrid  urine.  Zymase 
was  obtained  by  Biichner  through  the  pressing  of  the  ground  yeast 
cells,  as  has  been  described.  This  same  method  was  later  applied 
to  the  lactic  acid  bacteria  and  the  lactacidase  obtained. 

Oxidizing  Enzymes.— The  most  typical  example  of  an  oxidizing 
enzyme  is  the  vinegar  oxidase,  the  action  of  which  is  fairly  well 
known. '  The  reaction  may  be  written  in  the  simple  form 

CH3CH2OH     +     O2     =     CEbCOOH     +     H2O. 

Since,  however,  many  side  reactions  may  occur,  the  bacterial,  oxida- 
tion of  alcohol  is  not  in  reality  capable  of  so  simple  an  expression. 

Reducing  enzymes  are  the  most  common  of  ferments.  They  are 
formed  by  practically  all  plants  and  animals  and  contained  by  all 
but  a  very  few  bacteria,  Strept.  lacticus  being  one  of  the  few  excep- 
tions. In  this  case  the  absence  of  the  enzyme  is  used  as  a  diagnostic 
test  for  the  organism.  One  of  the  most  important  reductases  is 
the  peroxidase  which  reduces  hydrogen  peroxid  to  water  with  the 
liberation  of  oxygen. 

2H2O2     +     peroxidase      =     2H2O     +     O2. 

Others  which  reduce  nitrates  to  nitrites  of  particular  interest  to 
students  of  agriculture  are 

2KNO3      =     2KNO2     +     O2. 

Or  at  times  they  may  reduce  the  nitrite  to  elementary  nitrogen: 

2Ca(NO3)2      =     2CaO     +     2N2     +     5O2. 

Under  appropriate  conditions  the  important  element,  nitrogen, 
may  thus  be  lost  from  the  soil  by  denitrification.  In  a  similar  way 
sulphates  are  reduced  to  hydrogen  sulphid: 

H2S04     =     H2S     +     2O2. 

REFERENCES. 

Bayliss:     The  Nature  of  Enzyme  Action. 
Euler:     General  Chemistry  of  the  Enzymes. 
Falk:     The  Chemistry  of  Enzyme  Actions. 
Robertson:     The  Physical  Chemistry  of  the  Proteins. 


CHAPTER  VIII. 
BACTERIAL  METABOLISM  PRODUCTS. 

BACTERIA  are  able  to  bring  about  enormous  changes  in  their 
media  in  a  very  short  time.  This  is  due  in  no  small  measure  to  their 
method  of  metabolism  which  differs  from  that  of  the  animal,  in 
most  cases,  in  being  a  process  of  incomplete  oxidation,  whereas  that 
of  the  animal  is  a  process  of  complete  oxidation.  For  this  reason, 
many  of  the  organisms  of  especial  economic  importance  often  leave 
products  of  considerable  commercial  value. 

Physiologic  Classification.— From  a  physiologic  viewpoint  Jordan 
divides  the  substances  produced  by  bacterial  metabolism  into  four 
classes : 

1.  The  secretions,  or  those  substances  which  serve  some  purpose- 
ful end  in  the  cell  economy.    These  may  either  be  retained  within 
the  cell  or  may  pass  out  into  the  surrounding  medium. 

2.  The  excretions,  or  those  substances  that  are  ejected  because 
useless  to  the  organism;  the  ashes  of  cell  metabolism. 

3.  The  disintegration  products,  or  those  bodies  that  are  produced 
by  the  breaking  down  of  food  substances.    Their  nature  is  deter- 
mined partly  by  the  chemical  structure  of  the  nutrient  and  partly 
by  the  specific  bacteria  concerned  in  the  disintegration.  Some  of  the 
most  conspicuous,  if  not  the  most  important,  of  bacterial  products 
belong  to  this  class,  enzyme  activity  being  largely  responsible  for 
their  existence. 

4.  The  true  cell  substance.    To  this  class  belongs  the  cell  proto- 
plasm, those  products  which  are  being  built  up  into  cell  protoplasm, 
and  those  substances  which  are  being  broken  down  but  have  not 
yet  reached  the  stage  where  they  are  cast  off  from  the  cell. 

The  great  objection  which  may  be  brought  against  such  a  classi- 
fication is  that  although  many  products  can  be  definitely  placed, 
others,  for  instance  pigments,  cannot. 

Carbohydrate  Metabolism.— Products  from  carbohydrate  metab- 
olism vary  greatly,  depending  upon  the  species  of  bacteria,  age, 
medium,  and  whether  grown  in  the  presence  or  absence  of  oxygen. 
Some  writers  distinguish  six  types  of  microorganisms,  depending 
upon  the  change  which  they  produce  in  their  media,  namely: 

1.  Complete  oxidation  which  occurs  only  to  a  limited  extent 
among  bacteria  and  then  only  where  there  is  a  ready  supply  of 
oxygen,  as  is  the  case  in  a  well-aerated  soil  in  filters  or  on  the 
surface  of  decaying  substances. 

2.  Partial  oxidation  is  much  more  common  among  microorganisms 
than  is  complete  oxidation.    The  product  formed  is  also  often  of 
considerable  commercial  value.    This  is  the  case  in  the  oxidation 


CARBOHYDRATE  METABOLISM 


83 


of  alcohol  to  acetic  acid.  On  the  other  hand,  the  products  formed 
may  serve  as  food  to  other  microorganisms  and  thus  be  completely 
oxidized.  Acetic  acid,  if  not  too  strong,  may  be  further  oxidized  to 
carbon  dioxid  and  water,  as  sometimes  occurs,  resulting  in  a  decrease 
in  the  strength  of  vinegar. 

3.  Alcoholic  fermentation  is  brought  about  by  yeast;  yet  there 
are  bacteria  which  possess  the  power  of  producing  alcohol,  but  none 
of  them  are  of  economic  value.  Such  organisms  have  been  obtained 
from  hay  (B.  fitizianus)  and  sheep  manure  (B.  ethaceticus).  The 
Bad.  pneumonias  of  Friedlander  is  not  only  a  pathogenic  organism, 
but  also  possesses  the  power  of  decomposing  sugar  solutions  with  the 
formation  of  ethyl  alcohol  and  acetic  acid. 

The  reaction  as  brought  about  by  yeast  is  due  to  the  endo-enzyme, 
zymase,  first  isolated  by  Biichner.  The  reaction  is  dependent  upon 
a  readily  available  supply  of  phosphate,  and  according  to  Harden 
this  forms  an  intermediate  product  with  glucose,  thus: 


2C6Hi2O6 


2PO4HR2   -  2CO2 


I 

2C2H6O 


2H2O 


II 
2H2O 


C6Hi2O6  +  2PO4HR2 


According  to  equation  (I),  two  molecules  of  glucose  are  concerned 
in  the  change,  the  carbon  dioxid  and  alcohol  being  equal  in  weight 
to  one-half  of  the  sugar  used,  and  the  hexosephosphate  and  water 
representing  the  other  half.  In  the  second  equation  the  phosphate 
is  again  liberated,  and  the  hexose  presumably  fermented. 


I 

CHO 
CHOH 

CHOH 

I 
CHOH 

CHOH 

I 

CH2OH 
Glucose. 

IV 

Methylglyoxal. 
CHO 

CO 

CH3 

CHO 


H 
OH 


II 

CHO 

C(OH) 

II 
CH 

CHOH 
CHOH 

CH2OH 

Enol  form. 


VI 


H2O 


VII 
COOH 
CHOH 
CH3 


H 


CHO 


CHOH      OH  COH 

I  II 

CH2OH  CH2 
Glyceraldehyde. 


CHO 

->     CO 

CH3       + 

Methyl-glyoxal. 


H2O      + 


COOH 
CHOH 
CH3 


Lactic  acid. 


III 
CHO 

f 

CH2 

CHOH 
I 
CHOH  -* 

CH2OH 
Keto  form. 

VIII 
C02 
->  CH2OH 

CH3 
CO2 
->     CH2OH 

CH3 

Alcohol  and  carbon 
dioxid. 


84  BACTERIAL  METABOLISM  PRODUCTS 

Wohl  has  developed  a  theoretical  scheme  of  reactions  by  which 
the  process  of  alcoholic  fermentation  could  be  represented.  In  the 
first  place  the  elements  of  water  are  removed  from  the  oc  and  $ 
carbon  atoms  of  glucose  (I)  and  the  resulting  enol  (II)  undergoes 
conversion  into  the  corresponding  keton  (III),  which  has  the  consti- 
tution of  a  condensation  product  of  methylglyoxal  and  glyceral- 
dehyde,  and  hence  is  readily  resolved  by  hydrolysis  into  these  com- 
pounds (IV).  The  glyceraldehyde  passes  by  a  similar  series  of 
changes  (V,  VI)  into  methylglyoxal,  and  this  is  then  converted  by 
addition  of  water  into  lactic  acid  (VII),  a  reaction  common  to  all 
ketoaldehydes  of  this  kind.  Finally,  the  lactic  acid  is  split  up  into 
alcohol  and  carbon  dioxid  (VIII). 

In  alcoholic  fermentation  there  also  results  small  quantities 
(0.1  to  0.7  per  cent.)  of  fusel  oil.  This  contains  normal  propyl 
alcohol,  primary  isobutyl  alcohol,  primary  iso-amyl  alcohol,  and  the 
optically  active  (primary)  iso-amyl  alcohol.  It  was  thought  at 
one  time  that  these  resulted  from  the  fermentation  of  the  glucose, 
but  Ehrlich  in  a  series  of  masterly  researches,  shows  conclusively 
that  their  origin  is  the  ammo-acid  which  result  from  the  hydrolysis 
of  the  proteins,  the  reactions  of  which  may  be  given  as  follows  : 


CH—  CH2-rCHNH2—  COOH  +H2O^(CH3)2—  CHCH2CH2OH  +CO2  +NH3 
Leucine.  Primary  isobutyl  alcohol. 

II 

CH3CH(C2H5)—  CH(NH2)COOH  +  H2O  -+  CH3CHC2H5CH2OH  +  CO2  +  NH3 
Isoleucine.  Primary  iso-amyl  alcohol. 

Succinic  acid  also  occurs  among  the  products  resulting  from  alco- 
holic fermentation  of  sugar  and  has  its  origin  in  the  amino-acids.  It 
results  when  aspartic  acid  is  acted  upon  by  putrefactive  bacteria- 

COOH—  CHa—  CH—  NH2COOH  +  H2=COOH—  CH2—  CH2—  COOH  +  NH3 

This,  however,  differs  from  the  first  reaction  in  that  it  is  a  process 
of  partial  reduction  and  not  hydrolysis. 

Other  bacteria  have  been  studied  which  possess  the  power  of  pro- 
ducing butyl  and  amyl  alcohol  from  carbohydrates.  It  is  still  an 
.open  question  to  what  extent  the  amyl  alcohol  (fusel  oil)  produced 
during  an  impure  alcoholic  fermentation  is  due  to  bacteria,  for  it 
is  known  that  some  alcohol  yeasts  possess  the  power  of  decomposing 
two  protein  decomposition  products,  leucin  and  isoleucin,  with  the 
production  of  fusel  oil. 

4.  Acid  Production.—  In  general  it  may  be  stated  that  an  acid 
reaction  is  caused  by  the  fermentation  of  one  of  the  sugars,  glycerin, 
or  a  similar  substance  in  the  nutrient  media.  It  is  one  of  the  more 
constant  physiologic  characteristics  of  bacteria,  and  in  addition  to 


ACID  PRODUCTION  85 

being  of  considerable  economic  value  is  often  used  advantageously 
to  distinguish  closely  related  species,  notably  in  the  groups  of  para- 
typhoid and  dysenteria  bacilli.  In  addition  to  the  acid  produced 
by  the  fermentation  of  carbohydrates,  many  bacteria  hydrolyze 
proteins  with  the  formation  of  an  acid  reaction.  Formic  acid  is 
the  simplest  organic  acid  which  can-  be  formed.  It  is  produced 
by  B.  typhosus,  the  causative  agent  of  typhoid  fever.  B.  typhosus 
does  not  form  gas,  but  $.  coli  does;  and  in  this  latter  case  it  may 
result  from  the  decomposition  of  the  formic  acid. 

HCOOH     =     H2     +     CO2. 

Acetic  acid  is  one  of  the  most  important  acids  formed  by  bacteria. 
Bact.  aceti  and  Bact.  pasteurianum  are  two  of  the  more  important 
acetic  acid-forming  bacteria.  They  occur  in  fermenting  fruit  juices 
and  convert  the  alcohol  into  acetic  acid. 

CH3—  CH2—  OH     +     O2     =     CHsCOOH     +     H2O. 

Many  other  species  also  possess  the  power  of  transforming 
alcohol  and  other  substances  containing  the  characteristic  radical 
—  CH2CH2OH—  into  acetic  acid.  This  fermentation,  however,  can 
take  place  only  within  certain  limits  of  concentration,  and  even  then 
there  must  be  available  nitrogen  in  the  form  of  proteoses,  peptones 
or  amino-acids,  and  mineral  elements,  especially  phosphorus  in  the 
form  of  a  phosphate.  Acetic  acid  is  produced  on  the  commercial 
scale  by  a  number  of  processes.  Two  of  the  best  are  the  Orleans 
and  the  Quick,  or  German,  methods. 

Lactic  Acid.—  This  product  is  formed  by  a  great  number  of  bac- 
teria. The  chief  species,  however,  is  the  Streptococcus  lacticus 
which  produces  only  a  scanty  growth  on  agar,  but  an  excellent 
growth  in  milk,  bringing  about  a  solid  curdling  in  a  few  days.  The 
lactose  of  the  milk  is  first  inverted  forming  two  hexoses—  dextrose 
and  galactose. 


+     H2O      =     C6Hi2O6     +     C6Hi2O6 
Lactose.  Dextrose.  Galactose. 

The  hexose  in  turn  is  decomposed  yielding  two  molecules  of  lactic 
acid. 

C6Hi2O6      =     2C3H6O3 
Dextrose.  Lactic  acid. 

In  actual  experience  the  reaction  occurring  is  not  as  simple  as 
written  in  the  equation,  but  there  are  other  products  formed.  For 
instance,  B.  coli  ferments  glucose  with  the  formation  of  alcohol, 
carbon  dioxid,  hydrogen,  lactic  acid,  succinic  acid,  and  other 


86  BACTERIAL  METABOLISM  PRODUCTS 

products.     The  mechanism  of  the  fermentation  as   outlined  by 
Harden  is  illustrated  by  the  reaction. 

CH2OH  CH3CH2OH     +     3CO2     +     3H2 

CH  OH  CH2OH  COOH 

CH  OH  CH  OH  CH3  CH2 

II  I  I 
CHOHCHOH      =     CHOH     +     CH> 

III  \ 

CH  OH  CH  OH  COOH  COOH 


CHO      CH  OH 

CHO 
Glucose.  Lactic  acid.  Succinic  acid. 

Unless  some  base  be  added  to  neutralize  the  acid  as  formed,  the 
lactose  of  milk  is  never  completely  converted  into  lactic  acid  because 
the  accumulation  of  the  acid  is  sufficient  to  stop  most  bacterial 
growth,  as  is  seen  by  the  fact  that  meat  placed  in  buttermilk  will 
keep  for  some  time. 

Butyric  Acid.— Under  certain  conditions  a  further  decomposition 
of  the  lactic  acid  may  occur  with  the  formation  of  butyric  acid 
according  to  the  following  equation: 

2C3H6O3      =     C4H8O2     +     2CO2     +     2H2 
Lactic  acid.         Butyric  acid. 

However,  the  butyric  acid  bacteria  possess  the  power  of  ferment- 
ing sugar  with  the  formation  of  butyric  acid. 

C6H12O6     +     O2     =     CH3CH2CH2COOH     +     2CO2     +     2H2O 
Glucose.  Butyric  acid. 

Although  the  number  of  organisms  which  possess  the  power  of 
producing  butyric  acid  are  large,  they  are  not  as  numerous  as  those 
which  possess  the  power  of  forming  lactic  acid.  They  are  usually 
anaerobic  spore-bearers  with  a  tendency  to  form  spindle-shaped 
cells,  for  which  reason  they  have  been  given  the  name  Clostridium. 
Many  members  of  this  group  possess  the  power  of  fixing  nitrogen; 
they  probably  play  an  important  part  in  maintaining  the  nitrogen 
supply  of  the  soil.  B.  botulinus,  the  causative  agent  in  meat  poison- 
ing, forms  butyric  acid  as  does  also  B.  tetani. 

In  the  great  majority  of  cases  bacteria  produce  a  number  of  differ- 
ent products;  for  instance,  Azotobacter  chroococcum  produces  from 
the  carbohydrates,  ethyl  alcohol,  glycocoll,  acetic  acid,  butyric  acid, 
lactic  acid,  carbon  dioxid,  and  hydrogen.  The  quantity  and  quality 
of  the  different  products  vary  with  the  species  and  with  the  carbo- 
hydrate used. 

Bacterium  pasteurianum  grows  in  wine  and  cider  vinegars.  It 
produces  a  variety  of  products,  depending  upon  the  specific  sub- 
stance acted  upon.  It  produces  gluconic  acid  CH2OH  (CHOH)4- 
COOH  from  dextrose,  propionic  acid  (C2H5COOH)  from  propyl 


PRODUCTS  FROM  NITROGENOUS  COMPOUNDS  87 

alcohol  (C3H7OH),  and  acetic  acid  (CH3COOH)  from  ethyl  alcohol 
(C2H5OH). 

Other  Acid  Fermentation.—  In  addition  to  the  acids  described 
many  others  have  been  identified  among  the  products  of  carbohy- 
drate fermentation.  Many  molds,  especially,  possess  the  power  of 
fermenting  dextrose  with  the  formation  of  citric  acid,  the  general 
reaction  being  as  follows  : 

CH2COOH 

2C6Hi2O6     +     2O2      =     2HOC—  COOH  +     2H2O 

CH2COOH 
Dextrose.  Citric  acid. 

Patents  have  been  taken  out  on  this  method  and  attempts  made 
to  produce  citric  acid  on  a  commercial  scale,  but  so  far  without  any 
great  success. 

Oxalic  acid  is  also  produced  by  certain  molds  in  sugar  solutions' 
and  where  care  has  been  taken  to  neutralize  the  acid  so  formed 
one-half  the  calculated  theoretical  yield  has  been  obtained  from 
cane  sugar.     Formic  (H  COOH)  and  valeric  acids  (C4H9COOH) 
are  also  produced  by  some  microorganisms. 

Oxidation  of  Organic  Acids.—  The  organic  acids  formed  in  the 
various  processes  of  carbohydrate  fermentation  are  often  further 
oxidized  by  bacteria,  yeasts,  or  molds  to  simpler  products.  Ordi- 
narily the  process  consists  of  a  complete  oxidation.  This  may  be  the 
case  in  sauerkraut,  ensilage,  pickles,  etc.  Thus  O'idium  lactis  destroys 
the  lactic  acid  of  sour  milk  with  the  formation  of  carbon  dioxid 
and  water. 

+     3O2      =     3CO2     +     3H2O. 


At  times  the  spoiling  of  pickles  is  due  to  the  oxidation  of  the  acetic 
acid  by  yeasts  which  grow  in  a  thin  white  scum  over  the  surface. 

Fats.—  A.  comparatively  few  microorganisms  attack  fat.  When 
they  do  the  decomposition  of  the  fat  is  comparatively  simple.  One 
molecule  of  the  neutral  fat  is  split  with  the  formation  of  one  mole- 
cule of  glycerin  and  three  of  fatty  acids. 

H2COOC—  CisHai  H2COH 

HCOOC—  CisHsi     +     3H2O  H  COH     +     3Ci6H3i—  COOH 

H2COOC—  Ci8H3i  H2COH 

Neutral  fat.  Glycerin.  Fatty  acid. 

The  glycerin  is  readily  used  by  the  microorganism,  whereas  the 
fatty  acids  are  but  very  slowly  oxidized  and  that  by  only  a  few 
species. 

Products  from  Nitrogenous  Compounds.—  Proteins  are  complex 
organic  substances  composed  of  carbon,  hydrogen,  oxygen,  nitrogen, 


88  BACTERIAL  METABOLISM  PRODUCTS 

and  generally,  but  not  always,  sulphur,  and  sometimes  phosphorus. 
The  proportion  of  these  constituents  is  approximately  C,  50-55 
per  cent.;  H,  6-7.3  per  cent.;  0,  19-24  per  cent.;  N,  15-19  per  cent. 
S,  when  present,  0.3-2.5  per  cent.;  and  P,  0.4-0.8  per  cent.  They  are 
substances  which  in  the  main  consist  of  combinations  of  oc  amino- 
acids  or  their  derivatives.  The  decomposition  products  of  proteins 
include  proteoses,  peptones,  peptides,  carbon  dioxid,  ammonia, 
hydrogen  sulphid  and  amino-acids.  The  amino-acids  constitute  a 
long  list  of  important  substances  which  contain  nuclei  belonging 
either  to  the  aliphatic,  carbocylic,  or  heterocyclic  series.  The 
present  list  includes  glycocoll  (glycin)  alanin,  serin,  phenylalanin, 
tyrosein,  cystin,  tryptophan,  histidin,  valin,  argin,  leucin,  isoleucin, 
lysin,  aspartic  acid,  glutamic  acid,  prolin,  oxyprolin,  and  norleucin. 
Many,  especially  of  the  saprophytic  bacteria  which  occur  in 
the  soil,  have  the  power  of  breaking  down  native  proteins  with  the 
formation  of  the  various  amino-acids.  Undoubtedly  the  complex 
organic  compounds  which  are  being  isolated  from  the  soil,  and 
which  are  assumed  by  some  to  play  such  an  important  part  in  soil 
fertility,  have  just  such  an  origin.  But  it  is  usually  the  case  in  all 
media  that  the  bacterial  catabolism  carry  the  substance  much  farther 
than  the  amino-acid.  The  extent  of  this  change  varies  greatly  with 
the  species  of  microorganisms  and  the  conditions  under  which  they 
are  acting.  Kendall  summarizes  some  of  the  further  changes  which 
may  occur  as  follows: 

1.  The  reductive  deaminization  of  amino-acids  to  fatty  acids  with 
the  same  number  of  carbon  atoms. 

RCH2CHNH2COOH   +  H2   =  RCH2CH2COOH   +  NH3 

2.  Hydrolytic  deaminization  of  amino-acids  to  oxyacids  with  the 
same  number  of  carbon  atoms. 

R— GHz— CHNHz— COOH  +  H2O  =  R— GHz— CHOH— COOH  +  NH3 

3.  Oxidative  deaminization  of  amino-acids  to  keto-acids  with  the 
same  number  of  carbon  atoms. 

R— GHz— CHNHz— COOH   +  O2   =  R— GHz— CO— COOH   +  NH3 

The  above  reactions  may  be  taken  as  types  of  the  last  stages  of 
the  reactions  brought  about  by  the  ammonifying  bacteria  within 
the  soil. 

4.  Carboxylic  decomposition  of  amino-acid  to  amine  with  one 
less  carbon  atom. 

R— CHz— CH— NHz— COOH    =  R— GHz— CH2— NH2  +  CO2 

5.  Carboxylic  decomposition  with  the  formation  of  fatty  acids. 

R— GHz— CH2— COOH    =  R— GHz— CH3  +  CO2 


PRODUCTS  FROM  NITROGENOUS  COMPOUNDS 


89 


6.  Carboxylic  decomposition  with  the  formation  of  a  fatty  acid 
with  one  less  carbon  atom. 

R— CH2— CHzCOOH  +  3O    =  R— CH2— COOH  +  CO2  +  H2O 

Some  of  these  changes  are  often  produced  either  within  food  or  in 
the  alimentary  canal  and  are  of  considerable  clinical  significance. 
The  most  important  of  these  are  indol,  skatol  and  the  amins,  the 
simplest  of  which  is  trimethylamin. 

Indol  and  skatol  are  substances  produced  in  the  intestinal  tract 
from  tryptophan  chiefly  by  B.  coli  and  B.  proteus.  They  are  also 
formed  in  putrifying  proteins  and  it  is  to  indol  and  skatol  that  putri- 
fying  substances  owe  their  intensely  disagreeable  odor.  Indol 
gives  a  rose  color  with  nitrites  in  acid  solution  and  this  is  used  as  a 
method  of  identifying  certain  bacteria.  The  tryptophan  is  deam- 
inized  with  the  formation  of  indol  propionic  acid.  This  is  oxidized 
to  indol-acetic  acid.  From  this  latter  there  is  split  off  acetic  acid 
with  the  formation  of  indol.  The  reactions  are  as  follows: 

CH 


HC 

HC 

\ 


CH     CH— NH2 

/  I 

C          N  COOH 

H          H 
Tryptophan. 

CH 


HC 


HC 

X  C          N 
H          H 
Indol-propionic  acid. 

CH 


II         I 

CH     CH2 

COOH 


CH 


\ 


HC            C—      —  C— 

i              H                II 

-CH2 

HC 

i 

C  

II 

-CH 

II 

1              II                II 
HC            C               CH 

> 
COOH 

1 
HC 

II 
C 

II 
CH 

\      /\         / 

^ 

.   /\    , 

/ 

CH         NH 

CH         NH 

Indol-acetic  acid. 

Indol. 

Often  the  bacteria  split  out  carbon  dioxid  from  the  indol-acetic 
acid  with  the  formation  of  skatol : 

CH  CH 


HC 

I 

HC 


— CH2  HC 

II  I          -»         I 

CH     COOH        HC 


CH         NH 

Indol-acetic  acid. 


CH         NH 

Skatol. 


90  BACTEH1AL  METABOLISM  PRODUCTS 

These  substances  when  formed  in  the  intestinal  tract  are  absorbed 
and  carried  to  the  liver  where  they  are  conjugated  with  the  formation 
of  indican  which  is  then  eliminated  by  the  kidneys.    The  stages 
through  which  indol  passes  in  forming  indican  are  as  follows : 
H  H 

c  c 

</     \  /  \ 

HC     C CH  HC    C COH 

I    II     II   +  o  -   |   i|    .11    + 

HC     C      CH  HC    C      CH 

\   /\   /  \  /\   / 

CH    NH  C     NH 

H 
Indol.  Indoxyl. 

H  H 

C  C  O-SOsK 

/  \  /  \                   / 

HC        C-  -CO— SO3H  HC         C C 

H2S04        |  +     KOH         |                           |i 

HC        C  CH  HC        C               C— H 

\ /\       /  \ /\       / 
C          NH  C          NH 

H  H 

Indoxyl  sulphuric  acid.  Indican. 

Amins.— The  simplest  member  of  this  series  is  methylamin 
(CH3NH2)  which  is  produced  in  small  quantities  in  the  decomposi- 
tion of  nitrogenous  organic  matter.  It  occurs  in  herring  brine  along 
with  dimethylamin  (CH3)2NH  and  trimethylamin  (CH3)3N.  When 
alinin  is  acted  upon  by  the  carboxylase  the  carboxyl  group  of  the 
amino-acid  is  split  off  with  the  formation  of  ethylamin  according  to 
the  following  reaction : 

CH3CHNH2COOH      =     CH3CH2NH2     +     CO2 
Alanin.  Ethyl  amin. 

Others  of  special  interest  which  may  be  due  to  bacterial  activity 
are: 

1.  Cadaverin  from  lysin: 

CH2— CHzCHz— CH2— CH— COOH  CH2-^CH2CH2CH2CH2 

I  I  -         I  I 

NH2  NH2  NH2         -  NH2     +     CO2 

Lysin.  Cadaverin. 

2.  Putrescin  from  ornithin: 

CH2— CH2— CH2— CH— COOH  CH2— CH2— CH2— CH- 

I  I  -T*         I  I       +     C02 

NH2  NH2  NH2  NH2 

Ornithin.  Putrescin. 

3.  Beta-imidazole  ethylamin  from  histidin: 

HC NH\  H— C NH\ 

II  ^CH  \CH 

?~  ?~ 

CH2  CH2  +     CO2 

CHNH2  CH2NH2 

COOH 

Histidin.  Beta-imidazole  ethyl-amin. 


PRODUCTS  FROM  NITROGENOUS  COMPOUNDS  91 

Vaughan  considers  that  beta-imidazole-ethylamin  is  the  active 
principle  of  the  protein  molecule.  Some  of  these  amins  are  strong 
stimulants  of  the  heart  or  vasodilators.  It  is  quite  likely  that  their 
liberation  by  bacterial  activity  in  the  intestinal  tract  and  their 
subsequent  absorption  may  result  in  severe  constitutional  symptoms. 
These  compounds  belong  to  a  group  of  substances  called  "ptomains." 
They  are  alkaloid-like  bodies  of  basic  character  and  of  more  or  less 
well-known  structure.  Some  of  them  are  harmless,  while  others  are 
apparently  violent  poisons.  It  is  interesting  to  note  that  in  the 
majority  of  cases  the  poisonous  properties  decrease  or  at  times 
entirely  disappear  as  purification  proceeds  thus  indicating  that  the 
poisonous  principle  in  some  cases  at  least  is  an  impurity  associated 
with  them.  Their  production  is  not  limited  to  any  one  special  class 
of  bacteria,  for  Zinsser  defines  ptomains  as  "poisons  elaborated 
by  all  bacteria  that  are  capable  of  producing  protein  cleavage,  if 
planted  on  suitable  nutrient  materials  under  conditions  favoring 
growth.  The  matrix  of  these  poisons  is  the  protein  nutriment;  they 
are  not  products  of  intracellular  metabolism  specifically  characteris- 
tic of  the  bacteria  which  produce  them." 

Bacterial  toxins,  in  contradistinction  to  the  ptomains,  are  specific 
bacterial  poisons  which  are  characteristic  of  each  individual  species 
of  bacteria  and  are  truly  the  products  of  bacterial  metabolism  in 
that  they  emanate  from  the  cell  itself  either  as  a  secretion  or  excre- 
tion during  cell  life,  or  as  an  inherent  element  of  the  cytoplasm 
liberated  after  death. 

Enzymes  which  are  true  products  of  bacterial  metabolism  have 
been  considered  in  detail  in  the  preceding  chapter.  % 

Urea,  uric  acid,  and  hippuric  acid  are  the  forms  in  which  the 
waste  nitrogen  is  excreted  by  the  higher  animals.  There  are  a 
great  number  of  organisms  occurring  widely  distributed  which 
possess  the  power  of  changing  urea  into  ammonium  carbonate. 
This  is  a  simple  hydrolysis. 

NH2  NH40 

\  \ 

CO     +     2H2O      =  CO 

NH2  NH40 

Uric  acid  can  be  changed  in  several  ways  by  bacteria,  that  is,  it 
may  be  hydrolyzed  with  the  formation  of  dialuric  acid  and  urea. 

HN— C— O  HN— CO  NH2 

O    =  C    C— NHV  +  2H20         -*          OC     CHOH  +  CO 

>C  =     O  / 

HN-C— NH/  HN-CO  NH2 

Uric  acid.  Dialuric  acid.  Urea. 

On  oxidation  uric  acid  yields  various  substances,  alloxan,  urea, 
oxalic  acid,  carbonic  acid,  tartronic  acid,  allantoinic  and  uroxanic 


92 


BACTERIAL  METABOLISM  PRODUCTS 


acid.  If  uric  acid  is  given  to  man  the  greater  portion  of  it  is  prob- 
ably destroyed  by  bacteria  in  the  alimentary  tract,  but  the  liver  or 
kidneys  of  some  animals  secrete  a  uric  acid-destroying  enzyme  or 
uricolytic  enzyme,  called  uricase.  It  is  presumably  through  the 
formation  of  such  an  enzyme  that  bacteria  are  able  to  decompose 
uric  acid. 

Hippuric  acid  is  hydrolyzed  by  certain  bacteria  with  the  forma- 
tion of  benzoic  acid  and  glycocoll. 


HC 
HC 


C—  C—  NH—  CH^COOH 

\ 

CH 


CH 


COOH 


CH 


H20 


CH 


C 
H 

Hippuric  acid. 


HC 

| 
HC 

\    / 

C 

H 

Benzoic  acid. 


CH2NH2COOH 


Glycocoll 


The  glycocoll  may  then  be  deaminized  with  the  formation  of 
ammonia  and  acetic  acid.  Many  extremely  complex  transforma- 
tions of  organic  substances  occur  in  the  soil,  due  to  bacterial 
activity.  In  this  medium  many  of  the  changes  considered  above 
occur.  These  have  been  summarized  diagrammatically  for  the 
carbohydrates,  proteins,  oils,  and  waxes  by  Russell. 


Proteins. 


Carbohydrates; 

cellulose.         --  »   Oils. 


Amino-acids. 

1 

Acids.  | 

NH,     * 

I 

Other  compounds. 

4 

1 

1 

Hydroxy  acids- 

"Humus." 

Calcium 

Gaseous 

Nitrites. 

I 

salts. 

N. 

I 

Calcium  salts. 

CO,         |                   CO, 

Nitrates. 

T       \CO2 

CaCOi 

CaCO, 

Waxes. 


Undecom- 
posed. 


Products  from  mineral  compounds  may  be  either  oxidized  or 
reduced  by  bacteria.  Some  of  the  important  oxidations  are  the 
oxidation  of  ammonia  to  nitrites,  and  these  in  turn  to  nitrates. 


NH3        + 
HNO2     + 


3O 

O 


HNO2 


H2O 


These  changes  are  of  especial  interest  to  the  student  of  soils  and 
are  brought  about  by  the  nitrosomonas  and  nitromonas,  respectively. 

Ferrous  salts  may  be  oxidized  to  ferric,  while  sulfur  may  be 
oxidized  to  sulphuric  acid. 


3O 


VH2O      =     H2S04 


The  important  reduction  reactions  are  the  ones  which  occur  in 
denitrification  wherein  the  nitrate  is  changed  to  nitrite. 


Ca(NO3)2     =     Ca(NO2)2 


O2 


PRODUCTS  FROM  NITROGENOUS  COMPOUNDS  93 

Or  the  nitrate  may  be  completely  reduced  with  the  liberation  of 
gaseous  nitrogen,  thus  completely  removing  it  from  the  soil. 

2Ca(NO3)2      =     2CaO      +     2N2     +     5O2 

Sulphates  may  in  a  similar  manner  be  reduced  to  hydrogen  sulphid. 

H2S04  H2S     +     2O2 

Hence,  water  containing  calcium  sulphate  if  shut  off  from  air  may 
give  rise  to  that  ill-smelling  gas,  hydrogen  sulphid. 

Pigments.— Many  bacteria  produce  pigments,  among  which  are 
practically  all  the  colors  of  the  spectrum— violet,  indigo  blue  (B. 
molaceus,  B.  janthinus,  B.  cyanogenes,  B.  pyocyaneus),  green  (B. 
fluorescens) ,  yellow  (Staphylococcus  aureus,  Sarcina  lutea),  orange 
(Sarcina  aurantiacd),  and  red  (B.  prodigiosus) .  Usually  oxygen  is 
essential  to  the  production  of  pigments  and  their  intensity  varies, 
depending  upon  the  media  upon  which  the  organism  is  grown. 

The  phenomenon  of  pigment  production  has  long  attracted  the 
attention  of  bacteriologists,  and  many  attempts  have  been  made  to 
explain  their  occurrence;  but  so  far  none  of  the  explanations  would 
seem  to  be  wholly  satisfactory.  The  pigment  seems  to  be  of  no 
material  advantage  to  the  organism,  for  colorless  strains  may  be 
cultivated  which  possess  all  of  the  properties  of  the  original  strain 
with  the  exception  of  pigment  production.  There  is  no  evidence 
that  they  protect  the  organism  against  light,  nor  is  there  anything 
that  would  lead  to  the  belief  that  (analogous  to  hemoglobin)  they 
form  a  loose  combination  with  the  oxygen  which  under  certain 
circumstances  may  be  liberated.  The  pigment  does  not  make  it 
possible  for  the  organisms  to  assimilate  carbon  dioxid  as  does  the 
chlorophyll  of  the  higher  plants  in  the  majority  of  cases.  The  best 
evidence,  therefore,  points  to  the  conclusion  that  they  are  mere 
by-products  that  have  no  particular  meaning  to  the  organism. 

Beijerinck  divides  chromogenic  bacteria  into  three  classes: 

1.  Chromophorous  bacteria,  in  which  the  pigment  remains  within 
the  cell  and  has  a  certain  biological  significance  analogous  to  the 
chlorophyll  of  higher  plants.    To  this  class  belong  the  green  bacteria 
and  the  red  sulphur  bacteria,  or^purple  bacteria. 

2.  Chromoparous,  or  true  pigment-forming  bacteria,  which  set  free 
the  pigment  as  a  useless  excretion,  either  as  a  color-body  or  as  a 
leuco-body  which  becomes  colored  through  the  action  of  atmospheric 
oxygen.    The  cells  themselves  are  colorless  and  may  under  certain 
conditions  cease  to  produce  pigments.     To  this  class  belong  B. 
prodigios'ws  and  others. 

3.  Parachrome  bacteria  which  form  their  pigment  as  an  excretory 
product  but  retain  it  within  their  body,  as  B.  janthinus  and  others. 

The  chemical  nature  of  pigments  is  not  well  understood,  but  it  is 
known  that  they  differ  in  solubility  and  are  usually  classified 
according  to  solubility  in  water,  alcohol,  chloroform,  ether,  benzol, 


94  BACTERIAL  METABOLISM  PRODUCTS 

and  other  solvents.  The  pigment  produced  by  Azotobacter  chroo- 
coccum  is  insoluble  in  all  of  these  solvents  but  dissolves  in  alkalies 
undergoing  decomposition  with  the  formation  of  a  dark  brown 
solution. 

Heat.— Probably  all  bacteria  liberate  energy  as  heat  in  their 
metabolic  process  and  there  are  a  number  which  liberate  it  in  suffi- 
cient amount  perceptibly  to  change  the  temperature  of  the  media 
in  which  they  grow.  This  is  exemplified  in  the  heating  of  fermenting 
silage,  manure,  and  hay.  At  times  the  temperature  is  raised  to  the 
kindling  point  with  the  result  that  spontaneous  combustion  may 
occur  in  hay  and  grain  stacks.  Bacteria  generate  considerable  of 
the  heat,  but  other  chemical  processes  are  also  active. 


FIG.  14. — Photogenic  bacteria  colonies  on  a  plate  photographed  by  means  of  their 
own  light.     (Lafar.)     (Buchanan's  Household  Bacteriology.) 

Light.— Sometimes  one  sees  on  the  surface  of  decaying  wood, 
fish,  or  various  meats  a  bright  illuminated  surface  which  at  times 
may  be  sufficient  for  the  photographing  of  objects  in  an  otherwise 
dark  room.  This  is  due  to  the  growth  of  certain  light-producing 
bacteria.  Other  organisms  produce  a  beautiful  phosphorescence. 
The  organisms  producing  light  are  especially  prone  to  occur  in  saline 
waters  and  are  invariably  aerobes. 

REFERENCES. 
Marshall:     Microbiology. 
Taylor:     Digestion  and  Metabolism. 
Lafar:     Technical  Mycology. 
Kendall:     Bacteriology— General,  Pathological  and  Intestinal. 


CHAPTER  IX. 

INFLUENCE  OF  TEMPERATURE  AND  LIGHT  ON 
BACTERIA. 

TEMPERATURE  influences  life  phenomena  in  two  ways— chemi- 
cally and  physically.  Chemically,  heat  influences  powerfully  the 
reacting  velocity  within  the  cell  and  the  aggregate  condition  of  the 
molecules,  or  coagulation.  Physically,  temperature  influences  the 
viscosity  of  the  liquids  composing  the  cell. 

Temperature  and  Speed  of  Reaction.— According  to  the  law  of 
Van't  Hoff  and  Arrhenius,  a  chemical  reaction  is  increased  two  or 
more  times  its  original  speed  whenever  the  temperature  is  increased 
10°  C.  This  holds  good  for  the  reactions  in  living  organisms,  within 
certain  limits  of  temperature,  as  well  as  for  non-living,  as  may  be 
seen  from  the  following  table  given  by  Clausen  in  which  is  recorded 
the  number  of  milligrams  of  carbon  dioxid  produced  by  100  grams 
of  lupine  seeds  in  one  hour : 

Carbon  dioxid  Increase 

Temperature.  produced.  lor  10r  C. 

0°  7.27 

5  13.87 

10  18.11                  10.84 

15  34.37 

20  43.55                  25.44 

25  58.76 

30  85.00                  41.45 
35                .   100.00 

40  115.90                  30.90 

45  104.45 

50  46.20                  69.70 

55  17.70 

The  above  table  shows  that  for  temperatures  below  40°  C.  there 
is  a  general  increase  in  the  speed  of  the  reactions  with  increases  in 
temperature.  However,  at  higher  temperatures  the  amount  of 
carbon  dioxid  diminishes  rapidly  with  further  increase  in  tempera- 
ture. This  is  very  generally  observed  in  enzymatic  processes,  as  at 
temperatures  over  60°  C.  enzymes  are  rapidly  decomposed  and 
many  become  immediately  inactive  when  they  are  heated  up  to 
63°  to  65°  C.  This  may  be  due  to  the  fact  that  the  enzymes  them- 
selves undergo  hydrolysis  which  also  would  follow  the  temperature 
law  of  Van't  Hoff  and  Arrhenius.  Furthermore,  enzymes  are  prob- 
ably protein  and  would  undergo  heat  coagulation.  This  would 
reduce  the  reacting  areas  between  enzymes  and  fermentable  sub- 


96  INFLUENCE  OF   TEMPERATURE  ON  BACTERIA 

stance,  and  hence  decrease  proportionally  the  speed  of  the  catalyzed 
reaction. 

The  protoplasm  composing  the  bacterial  cell  consists  of  carbo- 
hydrates, lipins,  proteins,  and  ash  having  a  definite  structural 
arrangement  within  the  living  cell.  The  cellular  protoplasm  is, 
therefore,  a  colloid  existing  during  life  in  the  soluble  condition,  but 
when  heated  there  occurs  an  irreversible  reaction  with  the  forma- 
tion of  a  gel.  This  heat-coagulation  of  the  protein  is  explained  by 
Robertson  as  essentially  a  phenomenon  of  dehydration,  the  first 
stage  of  which  consists  of  internal  neutralization  through  the  loss 
of  the  elements  of  water  from  end-groups  ( —  NH2  and  —  COOH) , 
thus: 

H2N— RCOH— N— R— COOH      =     HN— R— COH— N— R— CO     +H2O 

1 1 

In  the  second  stage,  or  true  coagulation,  there  is  a  polymerization 
of  the  amino-acids  with  the  formation  of  the  irreversible  gel. 

2H2N—R— COH— N—R— COOH     =     H2N— R— COH— N— R— COH— N— 
R— COH— N—R— COOH     +     H2O 

Relation  to  Heat.— From  the  above  theoretical  consideration  we 
should  expect  to  find,  and  do  actually  find,  an  upper  temperature 
limit  at  which  all  organisms  cease  to  function.  This  upper  limit 
varies  considerably  with  the  species  of  bacteria  and  the  condition 
under  which  it  is  being  held.  B.  phosphorescens  will  not  grow  above 
37°  C.,  B.  tuberculosis  above  42°  C.,  B.  thermophilis  above  72°  C., 
and  Setchell  has  found  bacteria  living  in  the  water  of  hot  springs 
at  a  temperature  of  89°  C. 

This  great  variation  in  temperature  requirement  of  bacteria  has 
led  to  their  division  into  four  classes : 

1.  Thermophilic,  or  heat-loving  bacteria,  are  those  that  develop 
at  relatively  high  temperatures,  usually  above  45°  to  50°  C.    These 
organisms  occur  in  the  water  of  hot  springs,  in  decaying  piles  of 
compost  or  manure,  in  fermenting  ensilage,  in  the  intestinal  contents 
of  man  and  animals.     To  this  class  belong  the  non-motile  bacilli 
isolated  by  Miquel  from  the  Seine,  which  grew  rapidly  at  tem- 
peratures around  70°  C.,  as  does  also  the  so-called  " Mudedinus 
thermophiles,"  described  by  Tsiklinsky,  which  develop  readily  at 
temperatures  slightly  above  this.     Most  of  the  thermophiles  are 
spore-bearing  bacilli  of  little  or  no  practical  importance. 

2.  Psychrophilic  bacteria  are  those  which  grow  best  at  relatively 
low  temperatures,  usually  below  10°  C.    They  are  most  common  in 
cold  waters  such  as  those  of  springs,  wells,  the  depths  of  lakes  or 
oceans  and  the  soils  of  arctic  regions.    Forster  has  described  certain 
phosphorescent  bacteria,  which  he  isolated  from  sea  water  which 
grow  readily  at  10°  C.    Many  bacteria  of  the  soil  must  belong  to  this 
class,  as  Conn  and  Brown  have  repeatedly  shown  that  soil  bacteria 


RELATION  TO  HEAT 


97 


increase  in  number  near  the  freezing-point.  Some  bacteria  of  this 
type  probably  play  important  roles  in  soil  fertility  and  in  the  decay 
of  foods  in  cold  storage. 

3.  Mesophilic  bacteria  are  those  whose  optimum  temperature  is 
between  these  two  extremes.  They  comprise  the  great  group  of 
pathogenic  organisms  occurring  in  the  bodies  of  men  and  animals. 
To  this  class  also  belong  many  of  the  decay  and  putrefying  organisms 
found  in  the  soil.  All  of  the  more  important  bacteria  belong  to 
this  group. 

Three  temperature  limits  may  be  distinguished  for  bacterial 
growth:  (a)  Minimum,  the  lowest  temperature  at  which  bacterial 
growth  will  occur.  This  for  the  true  thermophiles  is  about  40°  C., 
for  some  pathogenic  29°  C.,  and  for  the  mesophilic  as  low  as  0°  Cv 
or  in  solutions  which  do  not  solidify  it  may  be  even  lower  than  this. 
(b)  Optimum,  that  of  most  luxuriant  growth.  This,  like  the  minimum 
temperature,  varies  greatly  with  the  species,  (c)  Maximum,  the 
highest  temperature  at  which  growth  and  multiplication  can  take, 
place.  This  may  be  a  few  or  many  degrees  above  the  optimum. 
For  the  thermophilic  it  may  be  as  high  as  89°  C.,  whereas  for  the 
pathogenic  bacteria  it  lies  between  40°  and  50°  C.  The  growth  of 
some  pathogenic  organisms  at  a  high  temperature  for  some  time 
causes  them  to  lose  their  virulence  or  disease-producing  power,  and 
is,  therefore,  made  use  of  in  the  preparation  of  vaccines. 

The  temperature  relations  are  seen  from  the  following  table 
reported  by  Fischer: 


Temperature. 

Q          • 

Minimum. 

Optimum. 

Maximum. 

Psychrophilic  bacteria    . 

0 

15-20 

30 

Many  water  bacteria. 

Mesophilic  bacteria  . 

15-25 

37 

43    . 

Pathogenic      bacteria 

and  others. 

Thermophilic  bacteria    . 

25-45 

50-55 

85 

Spore-bearing        bac- 

teria from  soil,  feces 

and  thermal  springs. 

The  growth  temperature  range  of  an  organism  is  the  number  of 
degrees  difference  between  the  minimum  and  maximum.  This  is 
very  small  with  some  bacteria  like  thegonococcus,  thepneumococcus, 
the  tubercle  bacillus,  and  others  which  are  highly  susceptible  to 
temperature  changes  and  have  the  power  of  growing  only  within 
limits  varying  but  a  few  degrees  from  the  optimum.  However, 
most  pathogenic  bacteria  may  grow  at  temperatures  ranging 
between  20°  C.  and  40°  C.  Others,  like  the  colon  bacilli  group,  the 
Bacillus  anthracis,  and  the  Spirillum  cholera  asiaticce,  may  develop 
at  temperatures  as  low  as  10°  C.  and  as  high  as  40°  C.  or  over.  The 
7 


INFLUENCE  OF  TEMPERATURE  ON  BACTERIA 


range  of  temperature  at  which  saprophytic  bacteria,  including  soil 
organisms,  may  develop  is  usually  a  far  wider  one.  These  points 
are  well  illustrated  by  the  following  table  taken  from  Fischer: 


Temperature. 

Difference  between  Mir 

mum  and  maximum. 

Minimum. 

Optimum. 

Maximum. 

B.  phosphorescens     . 

9 

20 

38 

29 

B.  fluorescens 

5 

20-25 

38 

33 

B.  subtilis       .... 

6 

30 

50 

44 

B.  anthracis    .... 

12 

37 

45 

33 

Vibrio  cholerse 

10 

37 

40 

30 

B.  diphtherias 

18 

33-37 

45 

27 

Mic.  gonorrheaa    . 

25 

37 

39 

14 

Bact.  tuberculosis 

30 

37 

42 

12 

B.  thermophilis    .      .      . 

40 

60 

80 

40 

The  fatal  temperature  may  be  even  somewhat  higher  than  this. 
It  will  vary  with  a  number  of  factors,  the  condition  of  the  organism 
playing  a  great  part.  For  instance,  Duclaux  found  that  certain 
bacilli  (Tyrothrix)  found  in  cheese  are  killed  in  one  minute  at  a 
temperature  of  from  80°  to  90°  C.,  whereas  for  the  spores  of  the  same 
bacillus  a  temperature  of  from  105°  C.  to  120°  C.  was  required. 

Duclaux  considers  it  erroneus  to  speak  of  a  definite  temperature 
as  a  fatal  one;  instead  he  considers  it  better  to  speak  of  it  as  deadly. 
This  is  due  to  the  fact  that  the  length  of  time  an  organism  is  exposed 
to  a  high  temperature  is  important.  This  is  illustrated  by  the  experi- 
ments of  Christen  on  the  spores  of  the  bacilli  of  the  soil  and  of  hay. 
The  spores  were  exposed  to  a  stream  of  steam  and  the  time  noted 
which  was  necessary  to  kill  the  spores  at  the  various  temperatures. 


Temperature. 

100° 
105-110 

115 
125-130 

135 

140 


Time  reqired  to  kill  spores, 
over  16  hours. 
2  to  4  hours. 
30  to  60  minutes. 
5  minutes  or  more. 
1  to  5  minutes. 
1  minute. 


Moist  heat  is  much  more  effective  as  a  germicide  than  is  dry 
heat.  The  probable  explanation  of  this  is  that  where  dry  heat  is 
applied  it  must  be  high  enough  to  decompose  the  organic  constit- 
uents of  the  cell,  the  proteinaceous  substance  being  in  the  form  of 
the  anhydride  which  can,  in  the  presence  of  moisture,  take  up  water 
according  to  the  following  equation: 

HN— R— COH— N— RCO     +     H2O      =     H2N— RCOH— N— R— COOH 


Two  or  more  molecules  of  this  hydrated  protein  would  then  con- 
dense with  the  formation  of  the  non-reversible  gel. 


2H2NRCOHNRCOOH 


=  H2N— R— COH— N— R  COH— N— R  COH  NR— 
COHNR— COOH  +  H2O 


THERMAL  DEATH  POINT 


99 


That  moisture  is  essential  for  coagulation  of  proteins  is  illustrated 
by  the  following  table  from  the  work  of  Hiss  and  Zinsser: 

Egg  albumen  in  dilute  aqueous  solution  coagulates  at    56°  C. 

with  25  per  cent,  water  coagulates  at  74°-80°  C. 

"    18        "  "  80°-90°  C 

6  145° C. 

Absolute  anhydrous  albumin,  according  to  Haas,  may  be  heated 
to  170°  C.  without  coagulation. 

Moreover,  moist  heat  is  much  more  penetrating  than  is  dry  heat. 
This  is  illustrated  by  an  experiment  carried  out  by  Koch  and  his 
associates.  Small  packages  of  garden  soil  were  wrapped  with  vary- 
ing thicknesses  of  linen  with  thermometers  so  placed  that  the  tem- 
perature under  a  definite  number  of  layers  could  be  determined. 
These  were  exposed  to  hot  air  and  steam  for  four  and  three  hours, 
respectively,  with  the  following  results: 


Temperatures. 

Time  of 
application. 

Temperatures  reached  within  thicknesses  of  linen. 

20  thick- 
nesses. 

40  thick- 
nesses. 

100  thicknesses. 

Hot  air 
Steam   . 

130°-140°  C. 
90°-105.3°C. 

4  hours 
3  hours 

86° 
101° 

72° 
101° 

Below     Incomplete 
70°          sterilization 
101.5°    Complete 
sterilization 

The  comparatively  low  specific  density  of  the  steam  enables  it  to 
displace  the  air  from  the  interior  of  materials.  Furthermore,  when 
the  steam  comes  in  contact  with  the  substance  to  be  sterilized  it 
condenses  with  a  liberation  of  heat.  This  in  the  case  of  water  vapor 
amounts  to  536.6  calories. 

Although  the  spores  of  certain  bacteria  of  the  soil  can  withstand 
live  steam  for  several  hours,  they  may  be  destroyed  in  a  few  minutes 
or  even  instantaneously  in  compressed  steam  ranging  in  temperature 
from  120°  to  140°  C. 

The  germicidal  action  of  the  great  majority  of  disinfectants  is 
due  to  a  chemical  reaction  taking  place  between  the  protoplasm  of 
the  bacterial  cell  and  the  germicide.  This  reaction  follows  the 
temperature  law  of  Van't  Hoff  and  Arrhenius.  Hence,  the  mere 
raising  of  the  temperature  a  few  degrees  of  a  sugar,  salt,  acid  or 
alkali  solution  makes  of  it  a  disinfectant. 

Thermal  Death  Point.— The  thermal  death  point  of  an  organism 
is  the  lowest  temperature  that  will  certainly  destroy  it  under  definite 
conditions.  These  conditions  are  time  (which  is  generally  taken  as 
ten  minutes),  amount  of  moisture  present,  the  reaction  and  com- 
position of  the  medium  in  which  the  organism  is  heated,  and  the 


100          INFLUENCE  OF  TEMPERATURE  ON  BACTERIA 

presence  or  absence  of  spores.  The  thermal  death  point  of  bacteria 
varies  witL  the  specific  character  of  the  organism  (some  organisms 
being  much  more  resistant  to  heat  than  are  others)  and  the  age  of 
the  culture,  young  cultures  being  more  resistant  than  older  cultures 
which  have  not  formed  spores,  especially  when  heated  in  the  prod- 
ucts resulting  from  their  metabolism. 

Cold.— It  has  been  shown  that  the  criterion  for  death  is  the  non- 
reversibility  of  the  change  brought  about  by  the  agency  in  question. 
Now,  does  the  lowering  of  the  temperature  bring  about  irreversible 
changes  in  the  protoplasm  as  does  the  raising  of  the  temperature? 
It  is  known  that  even  intense  cold  does  not  cause  these  irreversible 
reactions  in  proteins.  Where  death  does  occur  in  cold-blooded 
animals  and  in  plants,  it  must  be  due  to  the  formation  of  ice  crystals 
in  the  cells  which  may  mechanically  injure  and  kill  them.  This 
seems  to  be  the  case  in  the  freezing  of  plants.  Another  irreversible 
change  is  connected  with  the  thawing  of  the  cells  which  have  been 
frozen.  Barring  these  two  secondary  and  mechanical  complications, 
the  lowering  of  the  temperatures  does  not  seem  to  bring  about 
irreversible  changes  in  the  condition  of  the  protoplasm  which  are 
incompatible  with  life. 

When  the  temperature  of  the  protoplasm  becomes  sufficiently 
low,  for  example,  approximately  0°  C.,  the  velocity  of  the  chemical 
reaction  becomes  so  small  that  the  manifestations  of  life  cease.  The 
same  is  the  case  where  the  water  content  is  sufficiently  decreased. 
This  is  the  reason  why  seeds  of  higher  plants  and  spores  of  bacteria 
can  be  kept  alive  so  long.  Lack  of  water  may  reduce  the  reaction 
velocity  of  the  hydrolytic  processes  in  these  at  ordinary  tempera- 
ture to  such  an  extent  that  it  may  become  practically  zero.  So 
resistant  are  bacteria  to  low  temperature  that  they  may  be  frozen 
solid  and  kept  in  this  condition  for  days  and  even  weeks,  and  many 
survive.  Many  bacteria,  including  the  typhoid  and  colon  bacilli, 
will  survive  freezing  for  twenty-four  hours  in  liquid  hydrogen 
(—252°  C.)  and  develop  vigorously  when  brought  into  suitable 
media  at  an  optimum  temperature.  Bacteria  do  not  lose  their 
virulence  when  exposed  to  low  temperatures,  as  is  the  case  when 
exposed  to  comparatively  high  temperatures.  There  is,  however, 
a  tendency  for  the  number  of  organisms  gradually  to  decrease  as 
they  are  kept  in  the  frozen  condition.  When  typhoid  bacilli  are 
frozen  in  water,  approximately  90  per  cent,  of  them  die  during  the 
first  week,  95  per  cent,  succumb  by  the  end  of  four  weeks;  but  from 
four  to  six  months'  continuous  freezing  is  required  to  kill  all  of  the 
organisms.  The  speed  with  which  bacteria  disappear  from  a  frozen 
medium  varies  greatly  with  the  nature  of  the  medium.  It  is  yery 
slow  in  colloidal  substances  and  much  faster  in  crystalloids.  Alter- 
nate freezing  and  thawing  in  colloids  is  much  less  disastrous  to 
bacteria  than  the  same  treatment  in  aqueous  solutions.  It  is  prob- 


LIGHT-  /101 

able  that  the  crystals  formed  in  the  freezing  of  water  play  a  great 
part  in  the  mechanical  injuring  of  the  bacteria.  The  freezing  of  the 
soil  increases  not  only  the  number  of  bacteria  within  it,  but  the 
ammonifying  and  nitrogen-fixing  powers  of  the  soil.  Whether  or 
not  this  will  vary  with  the  water  content  of  the  soil  has  not  yet  been 
answered,  but  it  is  likely  that  as  the  moisture  content  increased 
the  greater  would  be  the  injurious  influence  of  the  low  temperatures. 
Light.— That  light  greatly  affects  the  metabolism  of  the  living 
cell  is  well  known.  However,  bacteria  are  even  more  sensitive  to 
light  than  are  most  cells.  Diffused  daylight  exerts  a  hindering  effect 


FIG.  15.— Thickly  sown  plate  culture  of  typhus  bacilli  on  agar-agar.  Covered 
with  paper  letters  and  exposed  to  the  sun's  rays  for  one  and  a  half  hours,  then  kept 
twenty-four  hours  in  the  dark,  whereupon  development  of  thickly  congregated 
whitish  colonies  was  found  only  at  the  parts  covered  by  letters.  (After  H.  Biichner.) 

upon  bacterial  growth  and  metabolism,  whereas  direct  sunlight  is 
highly  injurious  to  certain  bacteria,  many  microorganisms  being 
killed  almost  instantly  when  exposed  to  the  full  action  of  the  sun's 
rays.  The  different  colors  of  the  spectrum  do  not  act  alike.  The 
longer  rays,  from  red  to  green,  are  practically  without  influence 
upon  bacteria,  but  the  blue  and  violet  rays  have  the  most  marked 
germicidal  power. 

Since  light  has  no  effect  upon  bacteria  in  a  vacuum,  it  has  been 
inferred  that  the  changes  brought  about  in  the  bacterial  cell  are 
primarily  oxidation  changes  which  are  incompatible  with  the  life 
of  the  cell.  This  reaction  is  brought  about  more  rapidly  in  those 


102         INFLUENCE  Of    TEMPERATURE 'ON  BACTERIA 

cells  which  contain  considerable  fat,  and  this  may  be  the  reason 
why  some  spores  which  contain  an  oily  substance  are  especially 
sensitive  to  light.  Death  of  the  cell  in  many  cases  may  be  due  to  a 
type  of  coagulation  like  heat  coagulation,  for  Bovis  has  shown  that 
protein  solutions  in  quart  vessels  are  coagulated  by  exposure  to 
ultra-violent  light.  This  consists  of  two  stages— first,  that  of  dena- 
turation,  and  second,  agglutination,  or  flocculation. 


CHAPTER  X. 
EFFECT  OF  OTHER  AGENTS  ON  BACTERIA. 

Radium  Rays.— Fernan  and  Pauli  have  shown  that  the  exposure 
of  proteins  (serum  albumin)  in  acid  or  alkali  solution  to  radium 
radiations  causes  their  coagulation.  It  is  well  known  that  the  expos- 
ure of  living  tissue  to  these  rays  cause  their  destruction,  and  attempts 
have  been  made  to  treat  certain  bacterial  diseases  by  their  use,  but 
so  far  without  any  great  degree  of  success.  The  sterilization  of  milk 
and  other  foods  by  this  method  has  been  suggested,  but  its  practical 
application  appears  to  be  improbable  on  account  of  the  cost  and 
uncertainty  of  the  results. 

The  fixation  of  elementary  nitrogen  by  A.  chroococcum  is  dis- 
tinctly increased  when  the  air  is  activated  by  pitchblend,  somewhat 
better  results  being  obtained  with  weak  than  with  stronger  radio- 
active intensity.  Attempts  have  been  made  to  force  higher  plants 
by  its  use,  but  so  far  without  any  practical  success. 

Rontgen  Rays.— Although  rontgen  rays  are  used  in  the  treatment 
of  microbial  diseases  of  the  scalp  and  skin,  it  has  been  conclusively 
shown  that  they  are  not  even  inhibitory,  let  alone  fatal  to  the  cells. 
This  is  seen  from  the  results  by  Zeit,  who  found  that  bouillon 
and  hydrocell-fluid  cultures  in  test-tubes  of  non-resistant  forms  of 
bacteria  Was  not  killed  rontgen  rays  after  forty-eight  hours'  ex- 
posure at  a  distance  of  20  mm.  from  the  tube.  Tubercular  sputum 
exposed  to  these  rays  for  six  hours  at  a  distance  of  20  mm.  from  the 
tube  caused  acute  miliary  tuberculosis  of  guinea-pigs  inoculated 
with  it.  The  hopes  that  were  entertained  of  being  able  to  disinfect 
the  diseased  body  by  this  means  have  not  been  realized.  The  clinical 
results  which  are  sometimes  obtained  must  be  explained  by  factors 
other  than  their  direct  germicidal  influence,  possibly  by  the  pro- 
duction of  ozone,  hypochlorous  acid,  extensive  necrosis  of  the  deeper 
layers  of  the  skin  and  phagocytosis. 

Electricity.— The  influence  of  electricity  itself  upon  micro- 
organisms is  probably  very  slight,  but  it  is  often  difficult  nicely 
to  differentiate  between  purely  electrical  effects  and  chemical 
changes  which  are  produced  in  the  media  by  the  electric  current. 
A  direct  current  passing  through  a  nutrient  medium  will  cause  an 
electrolysis  which  is  usually  manifest  by  the  generation  of  acid  on 
the  positive  electrode  and  alkali  on  the  negative.  The  passing  of 


104  EFFECT  OF  OTHER  AGENTS  ON  BACTERIA 

an  electric  current  through  a  sodium  chlorid  solution  brings  about 
an  extremely  complex  change,  as  indicated  in  the  folio  wing  equations: 

2NaCl  =  2Na          +  C12 

2Na          +  2HOH      =  2NaOH  +  H2 

4C1           +  2HOH     =  4HC1       +  O2 

2NaOH    +  2C1           =  NaCIO     +  NaCl     +     H2O 

SNaCIO  =  NaClOs  +  2NaCl 

Many  of  the  products  so  formed,  if  in  sufficient  concentration,  are 
good  germicides  and  would  be,  therefore,  the  agents  causing  death 
instead  of  the  electricity  doing  it.  Moreover,  the  passing  of  an 
alternate  current  through  a  medium  may  heat  it  sufficiently  to  kill 
many  bacteria.  When  the  solution  is  properly  cooled  the  action 
of  the  current  is  practically  zero. 

Zeit,  who  has  made  a  very  careful  study  of  the  effect  of  electricity 
upon  bacteria,  summarizes  his  findings  as  follows : 

"1.  A  continuous  current  of  260  to  320  milliamperes  passed 
through  bouillon  cultures  kills  bacteria  of  low  thermal  death  points 
in  ten  minutes  by  the  production  of  heat  —98.5°  C.  The  anti- 
septics produced  by  electrolysis  during  this  time  are  not  sufficient 
to  prevent  growth  of  even  non-spore-bearing  bacteria.  The  effect 
is  a  purely  physical  one. 

"2.  A  continuous  current  of  48  milliamperes  passed  through 
bouillon  cultures  for  from  two  to  three  hours  does  not  kill  even 
non-resistant  forms  of  bacteria.  The  temperature  produced  by  such 
a  current  does  not  rise  above  37°  C.  and  the  electrolytic  products 
are  antiseptic  but  not  germicidal. 

"3.  A  continuous  current  of  100  milliamperes  passed  through 
bouillon  cultures  for  seventy-five  minutes  kills  all  non-resistant 
forms  of  bacteria  even  if  the  temperature  is  artificially  kept  below 
37°  C.  The  effect  is  due  to  the  formation  of  germicidal  electrolytic 
products  in  the  culture.  Anthrax  spores  are  killed  in  two  hours. 
Subtilis  spores  were  still  alive  after  the  current  was  passed  for  three 
hours. 

"4.  A  continuous  current  passed  through  bouillon  cultures  of 
bacteria  produces  a  strong  acid  reaction  at  the  positive  pole,  due 
to  the  liberation  of  chlorin  which  combines  with  oxygen  to  form 
hypochlorous  acid.  The  strongly  alkaline  reaction  of  the  bouillon 
culture  at  the  negative  pole  is  due  to  the  formation  of  sodium 
hydroxid  and  the  liberation  of  hydrogen  in  gas  bubbles.  With  a 
current  of  100  milliamperes  for  two  hours  it  required  8.82  mg.  of 
sulphuric  acid  to  neutralize  1  c.c.  of  the  culture  fluid  at  the  negative 
pole,  and  all  the  most  resistant  forms  of  bacteria  were  destroyed 
at  the  positive  pole,  including  anthrax  and  subtilis  spores.  At  the 
negative  pole  anthrax  spores  were  killed  also,  but  subtilis  spores 
remained  alive  for  four  hours. 


DRYING  105 

"5.  The  continuous  current  alone,  by  means  of  DuBois-Ray- 
mond's  method  of  non-polarizing  electrodes  and  exclusion  of  chemi- 
cal effects  by  ions  in  Kruger's  sense,  is  neither  bactericidal  not  anti- 
septic. The  apparent  antiseptic  effect  on  suspensions  of  bacteria 
is  due  to  electric  osmose.  The  continuous  electric  current  has  no 
bactericidal  nor  antiseptic  properties,  but  can  destroy  bacteria 
only  by  its  physical  effects— heat— or  chemical  effects— the  produc- 
tion of  bactericidal  substances  by  electrolysis. 

"6.  A  magnetic  field,  either  with  a  helix  of  wire  or  between  the 
poles  of  a  powerful  electromagnet  has  no  antiseptic  or  bactericidal 
effects  whatever. 

"7.  Alternating  currents  of  a  three-inch  Ruhmkorff  coil  passed 
through  bouillon  cultures  for  ten  hours  favor  growth  and  pigment 
production. 

"8.  High  frequency,  high  potential  currents— Tesla  currents- 
have  neither  antiseptic  nor  bactericidal  properties  when  passed 
around  a  bacterial  suspension  within  a  solenoid.  When  exposed  to 
the  brush  discharges,  ozone  is  produced  and  kills  the  bacteria." 

The  electric  current  is  used  in  the  purification  of  sewage,  the 
sterilization  of  milk,  the  improvement  of  wines,  and  the  purification 
of  water.  In  all  of  these  cases  the  effect  is  due  to  a  chemical  pro- 
duced by  the  electricity.  The  purification  of  water  is  due  to  the 
ozone  formed,  which  in  turn  acts  as  an  oxidizing  agent  toward  the 
bacteria.  Although  expensive,  it  is  one  of  the  most  effective  means 
of  rendering  water  safe. 

Drying.— The  results  which  have  been  reported  on  the  influence 
of  drying  upon  bacteria  are  exceedingly  divergent.  This  is  due 
mainly  to  the  fact  that  the  influence  exerted  by  drying  varies  with 
a  number  of  factors,  chief  among  which  are : 

1.  Light.— Bacteria  that  are  killed  in  a  few  minutes  in  direct 
sunlight  may  live  for  weeks  in  a  dark  place  or  even  in  diffused 
light. 

2.  Oxygen.— Pauli   and  his  associates  consider  death  through 
drying  as  due  to  an  oxidation  process.    They  found  that  bacteria 
die  much  faster  in  pure  oxygen  than  in  air.    Moreover,  they  found 
that  the  number  of  bacteria  dying  in  unit  time  under  constant  con- 
ditions is  proportional  to  the  number  surviving,  therefore,  com- 
parable with  the  simplest  chemical  processes,  the  monomolecular 
reactions. 

3.  Thickness  and   Nature  of  the  Medium   in  Which   They  Are 
Dried.— In  a  dried  medium  bacteria  usually  die  quickly  but  may 
survive  long  in  sputum  or  feces.    Moreover,  bacteria  suspended  in 
the  extract  from  a  rich  clay  loam  before  being  subjected  to  desicca- 
tion in  sand  live  longer  than  if  subjected  to  desiccation  after  sus- 
pensions in  a  physiological  salt  solution. 

4.  The  More  Complete  the  Drying  the  Shorter  the  Life.— Alternate 
drying  and  moistening  is  unfavorable. 


106  EFFECT  OF  OTHER  AGENTS  ON  BACTERIA 

5.  The  Higher  the  Temperature  the  Sooner  the  Bacteria  Perish.— 
Death  due  to  drying  is  probably  in  some  cases  due  to  a  non-reversible 
reaction  which  follows  the  well-known  temperature  law  of  Van't 
Hoff  and  Arrhenius.    In  other  cases  it  is  undoubtedly  due  to  the 
increased  osmotic  pressure  produced  by  the  removal  of  the  moisture. 

6.  Old  cultures,  unless  they  be  spore  bearers,  succumb  sooner  to 
drying  than  do  young  cultures. 

7.  The  influence  of  drying  upon  bacteria  varies  greatly  with  the 
species.  Whereas  the   gonococcus,    pneumococcus,    spirochete  of 
syphilis,  cholera  spirilla,  and  Pfeiffer  bacillus  can  withstand  drying 
only  a  few  hours,  the  typhoid,  diphtheria,  and  tubercle  bacilli  may 
survive  days;  and  tetanus,  anthrax,  and  many  soil  organisms  may 
survive  drying  for  months  or  even  years.    Ammonifying,  nitrifying, 
and  nitrogen-fixing  bacteria  have  been  isolated  in  great  numbers 
from  soils  which  have  been  kept  in  tight  bottles  air-dry  for  more  than 
fifty  years.    Even  the  non-spore-forming  types  of  Azotobacter  will 
withstand  desiccation  over  sulphuric  acid  for  a  considerable  time. 

Osmotic  Pressure.— Bacteria  vary  greatly  in  their  ability  to  with- 
stand great  osmotic  changes.  Some  are  quickly  plasmolyzed  in 
solutions  having  low  osmotic  pressure,  whereas  others  can  grow  in 
strong  sugar  or  salt  solutions.  This  factor  plays  a  great  part  in  the 
preserving  of  fruits  by  means  of  sugar,  of  pickles  and  cabbage  by 
means  of  salt,  and  many  fruits  by  drying.  Those  fruits  which  have 
the  highest  carbohydrate  content,  such  as  grapes  and  prunes,  are 
especially  easy  to  preserve  by  drying. 


FIG.  16.— Plasmolysis  of  various  bacterial  cells.     (Buchanan's  Household 
Bacteriology.) 

Probably  the  great  osmotic  pressure  in  the  soil  solution  of  alkali 
soils  plays  a  great  part  in  retarding  the  bacterial  activity  of  these 
soils.  In  this  case,  however,  there  is  also  a  physiological  factor  in 
which  the  living  protoplasm  of  the  cell  is  so  changed  in  its  chemical 
and  physical  properties  that  it  cannot  function  normally.  It  is 
found  that  equivalent  osmotic  concentrations  of  sodium  and  potas- 
sium salts  act  very  differently  upon  some  bacteria. 

Pressure.— Bridgman  found  that  the  application  of  very  great 
hydrostatic  pressure  resulted  in  the  coagulation  of  white  of  egg. 


SHAKING  107 

lie  applied  the  pressure  very  slowly  to  avoid  any  rise  in  temperature 
due  to  the  compression.  That  the  effect  is  not  due  to  heat  is  further 
demonstrated  by  the  fact  that  it  is  more  easily  obtained  at  0°  C. 
than  at  20°  C.  The  application  of  five  thousand  atmospheres  pro- 
duces stiffening  of  the  white  of  egg;  six  thousand  atmospheres 
applied,  for  thirty  minutes,  produced  an  appearance  of  the  white 
resembling  that  of  curdled  milk;  and  seven  thousand  atmospheres' 
pressure  brought  about  complete  gelatination. 

These  facts  seem  to  indicate  that  high  pressure  is  fatal  to  many 
bacteria.  Experiments  have  shown  this  to  be  the  case.  B.  anihracis, 
B.  pseudodiphtherice ,  M .  pyogenes,  var.  aureus,  and  O'idium  lactis 
survived  after  being  subjected  to  a  pressure  of  2000  atmospheres  for 
ninety-six  hours.  The  pigment  production  and  virulence  of  patho- 
genic organisms  were  either  diminished  or  completely  lost  after  such 
treatment. 

Successful  attempts  have  been  made  to  preserve  fruit  and  vege- 
tables by  exposing  them  to  high  pressure.  Apple  juice  subjected 
to  4000  to  6000  atmospheres'  pressure  for  thirty  minutes  did  not 
later  develop  gas.  Peaches  and  pears  exposed  to  this  pressure  did 
not  spoil  for  five  years.  Those  vegetables  on  which  are  found  resist- 
ant spores  could  not  be  preserved  by  such  pressures.  It  therefore 
appears  that  pressure  high  enough  for  the  coagulation  of  the  proteins 
is  fatal  to  the  less  resistant  bacteria. 

The  power  of  resisting  and  actually  functioning  under  high 
pressure  is  especially  necessary  for  the  denitrifying  bacteria  which 
live  at  the  bottom  of  the  ocean  and  return  to  the  atmosphere  the 
thousands  of  tons  of  combined  nitrogen  which  is  carried  each  year 
to  the  ocean  from  the  soil  and  in  the  sewers. 

Shaking.— It  is  well  known  that  proteins  may  be  coagulated  by 
shaking  and  that  proteolytic  enzymes  undergo  important  modifica- 
tions under  the  influence  of  shaking.  An  active  solution  of  proteo- 
lytic enzyme  introduced  into  a  reaction  tube  and  agitated  for  two 
minutes  may  lose  as  much  as  75  per  cent,  of  its  activity.  After  five 
minutes  the  disappearance  is  almost  total.  The  effect  of  shaking 
varies  with  the  speed,  temperature,  and  reaction  of  the  medium  in 
which  the  ferment  is  placed.  This  phenomenon  is  known  to  be  due 
to  a  coagulation  or  absorption  of  the  substance,  and  it  is  quite 
possible  that  part  of  the  influence  exerted  by  shaking  upon  bacteria 
is  due.  to  this  factor.  It  is  known,  however,  that  bacteria  may  be 
broken  into  the  finest  particles  by  the  rapid  shaking  of  cultures 
causing  death  at  times  by  a  disintegration  of  the  cell  body. 


CHAPTER  XL 
EFFECT  OF  CHEMICALS  ON  BACTERIA. 

Chemotaxis.— It  has  been  repeatedly  demonstrated  that  bacteria, 
like  other  free-moving  organisms,  are  apparently  attracted  by  cer- 
tain chemical  substances  in  solution  (positive  chemotaxis),  and 
repelled  by  others  (negative  chemotaxis) . 

Pfeiffer,  who  was  the  first  to  study  this  phenomenon,  developed  a 
very  simple  and  efficient  method  of  studying  it  with  bacteria.  A 
capillary  tube,  sealed  at  one  end  and  from  5  to  10  mm.  long,  is 
filled  with  a  5  per  cent,  slightly  alkaline  solution  of  Liebig's  beef 
extract  or  of  peptone.  The  outer  surface  of  the  glass  is  carefully 
cleaned  from  any  traces  of  the  bouillon  and  is  placed  in  a  drop  of 
water  containing  bacteria.  In  a  few  seconds  the  bacteria  are  found 
to  thickly  congregate  around  the  open  end  of  the  capillary  tube. 
According  to  the  view  held  by  Jennings,  the  swarming  of  bacteria 
around  any  point,  where  favorable  nutrient  conditions  exist  is  not  to 
be  looked  upon  as  due  to  a  definite  attraction  exerted  upon  the 
bacterial  cell,  but  as  caused  simply  by  the  tendency  to  remain  at 
those  points  where  the  conditions  are  favorable.  But  this  does  not 
seem  to  be  the  true  explanation,  for  had  the  capillary  tube  been 
filled  with  sugar,  or  glycerin,  which  are  the  best  foodstuffs  and  richest 
sources  of  energy,  th,ere  would  have  been  no  such  gathering  of  the 
the  bacteria  at  the  end  of  the  tube.  Moreover,  a  solution  of  0.019 
per  cent,  potassium  chlorid  plus  0.01  per  cent,  mercuric  chlorid 
attracts  bacteria  by  reason  of  the  potassium  which  it  contains,  but 
they  rush  into  the  tube  only  to  meet  their  death  from  the  mercury 
salt. 

The  explanation  given  by  Loeb  seems  to  be  more  reasonable: 
"Theoretically,  we  may  assume  that  if  substances  diffuse  in  air  or 
in  water,  the  particles  move  in  a  straight  line  away  from  the  center 
of  diffusion.  If  they  strike  an  organism  whose  surface  is  affected 
by  the  diffusing  substance  on  one  side  only,  the  contractile  proto- 
plasm, or  the  muscles,  turning  the  tip  or  the  head  of  the  whole 
organism  toward  that  side,  are  thrown  into  a  different  state  of  con- 
traction from  their  antagonists.  The  consequence  is  a  turning  or 
binding  of  the  tip  of  the  head  until  symmetrical  points  of  the  chem- 
ically sensitive  surface  of  the  body  are  struck  by  the  line  of  dif- 
fusion (or  the  diffusing  particles)  at  the  same  angle.  As  soon  as 
this  occurs  the  contractile  elements  on  both  sides  of  the  organ  or 
organisms  are  in  an  equal  state  of  contraction,  and  the  animal  will 


CHEMOTAXIS 


109 


bend  or  move  in  the  direction  of  the  lines  of  diffusion."  Why  one 
substance  should  act  positive  and  another  negative  is  at  present 
quite  inexplicable. 

Chemotaxis  can  take  place  only  in  media  which  permit  free 
movement  and  its  sphere  of  action  is  comparatively  small.  Different 
kinds  of  bacteria  by  no  means  react  in  the  same  way  to  the  same 
substance.  Furthermore,  the  action,  whether  positive  or  negative, 
chemotaxis  or  neutral,  varies  with  the  chemical.  The  salts  of  potas- 
sium are  among  the  more  active  positive  chemo tactic  substances, 
followed  by  sodium  and  rubidium.  The  alkaline  earths  are  less 
effective.  The  influence  of  a  salt  is  attributed  mainly  to  its  electro- 
positive constituent;  asparagin  and  peptone  are  strongly  chemo- 
tactic,  whereas  sugar  and  glycerin  are  inactive. 

Negative  chemotaxis  is  noted  when  capillary  tubes  are  filled 
with  free  acids  and  alkalies  or  with  alcohol.  In  some  salts  the  action 
of  the  acid  radical  and  that  of  the  base  neutralize  each  other 
(ammonium  carbonate  and  monobasic  potassium  phosphate).  In 
this  case  the  bacteria  are  neither  attracted  nor  repelled  by  the  sub- 
stance. 


FIG.  17.— Oxygen-loving  bacteria  infesting  a  thread  of  alga  lying  in  the  micro- 
spectrum.  The  chlorophyll  granules  contained  in  the  alga  cells  are  not  shown,  but 
the  spectrum  lines  are  given  to  denote  the  position  of  the  spectrum.  Mag.  200.  (After 
Engelmann.) 

Engelmann  ingeniously  made  use  of  this  phenomenon  as  a  test 
for  oxygen  and  the  effect  exerted  upon  assimilation  by  the  different 
parts  of  the  solar  spectrum.  If  a  thread  of  algae  and  some  aerobic 
bacteria  are  placed  under  an  air-tight  cover-glass,  the  bacteria  are 
active;  but  if  the  preparation  is  kept  in  the  dark  the  action  of  the 
bacteria  will  cease,  showing  that  all  the  oxygen  has  been  consumed. 
If  brought  back  to  the  light  as  the  algse  assimilate  carbon  dioxid 
with  the  elimination  of  oxygen  the  bacteria  again  become  active. 
If  exposed  to  the  spectrum  the  greatest  aggregation  of  bacteria 
occurs  at  the  red  end  of  the  spectrum,  indicating  that  the  maximum 
assimilative  activity  of  the  algse  protoplasm  is  proceeding  at  this 


110  EFFECT  OF  CHEMICALS  ON  BACTERIA 

point.    By  means  of  this  highly  sensitive  test  as  little  as  one-billionth 
part  of  a  milligram  of  oxygen  may  be  detected. 

It  is  quite  possible  that  the  phagocytes  which  play  such  a  part 
in  freeing  the  body  of  bacteria  are  directed  or  guided  in  their  choice 
and  perception  by  chemotaxis  to  the  bodies  which  they  ingest. 
The  attraction  of  leukocytes  toward  the  point  of  bacterial  invasion 
is,  in  part  at  least,  due  to  the  properties  of  the  bacterial  proteins. 
This  attraction  is  sometimes  increased  by  injecting  into  the  tissues 
at  the  point  of  infection  some  bland  substance,  such  for  instance  as 
bismuth  subnitrate. 

Disinfectants.— Of  great  interest  are  those  substances  which  in 
minute  quantities  destroy  the  life  of  the  cell.  These  substances 
when  considered  in  their  effects  upon  man  and  animals  are  called 
poisons.  But  when  considered  from  the  standpoint  of  micro- 
organisms they  are  called  germicides.  Analogous  with  the  general 
term  germicide,  are  the  terms  bactericide  and  fungicide.  A  disin- 
fectant is  a  substance  which  destroys  the  causative  agent  of  infec- 
tion. Although  disinfection  may  occasionally  mean  sterilization,  in 
the  majority  of  cases  it  does  not.  It  implies  the  destruction  of  those 
minute  forms  of  life  which  cause  disease. 

Antiseptics  prevent  decomposition  and  decay.  They  do  not 
necessarily  destroy  microorganisms;  they  prevent  their  growth  and 
activity.  One  and  the  same  substance  may  be  a  disinfectant  under 
one  condition  and  an  antiseptic  under  another.  Formalin  in  the 
proportion  of  1  to  50,000  is  an  antiseptic,  whereas  it  requires  from 
3  to  10  per  cent,  solution  to  be  a  disinfectant  in  a  reasonably  short 
time.  Mercuric  bichlorid  in  the  proportion  of  1  to  300,000  will 
sometimes  prevent  the  germination  of  anthrax  spores.  Yet  it 
requires  a  1  to  1000  solution  to  kill  them. 

The  term  preservative  is  usually  applied  to  those  substances 
which  are  added  to  foods,  feeding-stuff,  and  substances  of  similar 
origin  with  the  intention  of  preventing  decomposition  or  decay. 
These  may  be  either  comparatively  poisonous— benzoic  acid,  boric 
acid,  salicylic  acid,  formalin,  or  sulphates— or  the  non-poisonous 
substances— common  salt  or  sugar.  The  method  of  action  of  the 
two  is  markedly  different,  the  first  combining  with  the  protoplasm 
of  the  cell,  the  second  acting  through  increased  osmotic  pressure. 

Deodorants  are  substances  which  have  the  power  of  destroying 
or  masking  unpleasant  odors  arising  from  putrifying  or  fermenting 
organic  matter.  Deodorants  destroy  odors,  disinfectants  destroy 
germs.  A  deodorant  may  or  may  not  be  a  disinfectant.  Formalin 
is  a  good  disinfectant  and  deodorant,  whereas  charcoal  is  a  good 
deodorant  but  has  no  value  as  a  disinfectant. 

The  classification  of  disinfectants  is  difficult,  inasmuch  as  we  do 
not  understand  in  many  cases  their  complete  mode  of  action. 
Moreover,  almost  any  compound,  if  used  in  sufficient  concentration, 
may  act  as  an  antiseptic  if  not  as  a  disinfectant*  The  methods 


DISINFECTANTS  111 

most  often  used  in  classification  are  according  to  either  composition 
or  mode  of  action.  The  simplest  method  is  by  chemical  structures 
and  qualities  under  which  are  distinguished  the  following  natural 
groups:  acids,  alkalies,  metallic  salts,  hydrocarbons,  alcohols, 
aldehyds,  anesthetics,  essential  oils,  and  oxidizing  and  reducing 
agents.  The  first  three— acids,  alkalies,  and  salts— are  distinguished 
from  the  rest  by  being  electrolytes.  The  strength  of  acids  and  alka- 
lies is  dependent  upon  the  hydrogen  or  hydroxyl  ion  concentration 
with  the  metallic  salt;  the  action  is  dependent  upon  the  nature  of 
the  metallic  ion  and  the  degree  of  electrolytic  dissociation. 

Rosenau  classified  disinfectants  according  to  mode  of  action  as 
follows :  (1)  Those  compounds  which  destroy  by  oxidation,  as  ozone, 
chlorinated  lime,  potassium  permanganate,  and  the  halogens. 
(2)  The  destruction  by  ionic  poison  with  coagulation,  as  the  metallic 
salts,  mercury,  and  lead  salts.  (3)  Destruction  by  coagulation  and 
poisoning  not  ionic  in  character,  as  carbolic  acid  and  its  derivatives. 
(4)  Destruction  by  emulsoid  action,  that  is,  through  Brownian 
movement  and  adsorption;  soap  solutions  and  creolin. 

Laws  Governing  the  Action  of  Disinfectants.— These  have  been 
mainly  worked  out  by  Chick  who  found  that  disinfection  is  an 
orderly  time-process,  which  may  be  considered  analogous  with  a 
chemical  reaction,  viz.,  a  reaction  between  the  bacterium  on  the 
one  hand  and  the  disinfectant  on  the  other.  In  the  ideal  case  disin- 
fection proceeds  in  accordance  with  some  rule  analogous  to  the  mass 
law,  so  that  if  the  disinfectant  is  present  in  large  excess,  disinfection 
rate  at  any  moment  is  proportional  to  the  concentration  of  bacteria 

(—  -~  =  Kn,  where  n  is  the  concentration  of  bacteria  at  the  time  t, 

and  7v  is  a  constant,  depending  on  the  temperature  concentration 
of  disinfectant,  etc.). 

The  velocity  of  disinfection  increases  with  rise  in  temperature  in 
an  orderly  manner  according  to  the  well-known  equation  of  Arrhe- 
nius.  Some  idea  of  the  magnitude  of  the  effect  of  temperature  may 
be  gained  from  the  fact  that  with  metallic  salts  the  mean  velocity 
of  disinfection  increases  two-  to  four-fold  for  a  rise  in  temperature 
of  10°  C.,  whereas  with  phenol  it  was  as  high  as  eight-fold,  using 
B.  paratyphosus  as  the  test  organism  in  each  case.  Hence,  the  use 
of  a  disinfectant  at  a  comparatively  high  temperature,  other  things 
being  equal,  is  more  effective  than  its  use  at  a  low  temperature.  In 
reality,  a  solution  which  at  one  temperature  is  only  an  antiseptic 
may  become  a  disinfectant  by  a  small  increase  in  temperature. 

The  efficiency  of  a  disinfectant  varies  with  the  moisture.  A  dry 
poison  has  but  slight  action  on  microorganisms.  For  this  reason, 
dry  formaldehyde  gas  is  practically  without  effect.  In  a  similar 
manner  absolute  alcohol  has  not  nearly  the  same  germicidal  power 
as  has  50  to  70  per  cent,  alcohol.  This  is  probably  due  to  the 
absolute  alcohol  coagulating  the  outer  membrane  of  the  organism 


112 


EFFECT  OF  CHEMICALS  ON  BACTERIA 


and  thus  prevents  the  poison  from  diffusing  into  the  vital  part. 
The  burning  of  sulphur  in  a  dry  atmosphere  has  little  if  any  effect 
upon  bacteria,  but  in  the  presence  of  moisture  there  is  formed 
sulphurous  acid  which  is  a  rather  efficient  disinfectant. 

The  germicidal  property  of  salts  of  the  heavy  metals,  acids,  and 
alkalies  is  governed  in  a  large  measure  by  the  degree  of  ionization. 
Mercuric  chlorid  in  water  is  a  good  disinfectant,  but  in  alcohol  has 
little  or  practically  no  germicidal  properties.  The  addition  of  sodium 
chlorid  to  mercuric  chlorid  increases  the  solubility  of  the  latter  and 
yet  decreases  its  germicidal  power.  This  is  due  to  the  fact  that  there 
is  formed  a  double  salt: 

2NaCl     +     HgCl2      =     Na2HgCl4 

+  + 

This  is  poorly  dissociated  by  steps  into  Na  +  NaHgCU,  Na  -f 


HgCl4,  Hg  +  4C1.    The  number  of  Hg  ions  formed  is  very  small, 
therefore,  in  the  presence  of  sodium  chlorid. 

As  a  general  rule  the  addition  of  a  common  negative  ion  decreases 
the  number  of  ions  of  the  metal  going  into  solution.  If  mercuric 
chlorid  is  shaken  with  water,  the  salt  dissolves  until  there  is  an 
equilibrium  between  the  solid  phase  and  the  undissociated  molecules 
in  solution.  As  the  molecules  dissociate,  the  equilibrium  is  dis- 
turbed and  more  of  the  solid  dissolves  to  restore  it,  until  a  second 
equilibrium  is  established  between  the  ions  and  the  molecules. 
These  equilibria  may  be  expressed  by  the  equation 


[Hg"j  x 


[HgCl2] 


a  constant; 


since  the  concentration  of  the  undissociated  molecules  is  constant. 
So  long  as  there  is  any  undissolved  salt,  the  equation  becomes 


Hg     X    Cl 


a  constant. 


If  we  add  NaCl,  the  Cl  will  increase  the  concentration  of  the  Cl 
ions  which  will  combine  with  the  Hg,  giving  HgCl2,  which  will 
crystallize  out.  Moreover,  in  this  case  the  NaCl  combines  with  the 
HgCl2  giving  the  Na2HgCl4  which  greatly  decreases  the  Hg  ions  in 
solution.  The  effect  of  this  on  the  disinfecting  power  of  different 
dissociated  salts  of  mercury  on  anthrax  spores  is  indicated  in  the 
following  from  Paul  and  Kronig: 


Salt. 

Concentration. 

Colonies  after  20 
minutes'  exposure. 

Colonies  after  85 
minutes'  exposure. 

Colonies  after  90 
minutes'  exposure. 

HgCl2  .      . 

1/64  mol. 

7 

0 

0 

HgBr2 

1/64  mol. 

34 

0 

0 

HgCn2       . 

1/16  mol. 

0 

33 

0      . 

K2HgCl4    . 

1/1  6  mol. 

0 

K2HgBr4    . 

1/16  mol. 

5 

K2HgI4      . 

1/16  mol. 

389 

K2HgCn4  . 

1/16  mol. 

1035 

DISINFECTANTS 


113 


The  power  of  a  disinfectant  to  kill  bacteria  is  dependent  in  a 
remarkable  degree  upon  the  nature  of  the  medium  in  which  bacteria 
are  present  when  the  germicide  is  applied.  Almost  invariably  the 
greatest  germicidal  activity  is  shown  when  the  substance  acts  upon 
the  bacteria  freed  from  all  contaminating  culture  media  and  sus- 
pended in  distilled  water  or  salt  solution.  The  presence  of  proteins, 
peptones,  and  similar  substances  usually  cause  a  great  reduction  in 
the  germicidal  powers  of  the  substance.  This  is  also  the  case  in  the 
presence  of  pus,  many  of  the  organisms  being  partly  digested  in 
the  body  of  dead  leukocytes.  This  property  is  illustrated  by  the 
following  table  reported  by  Dakin  and  Dunham.  The  -  -  sign 
indicates  sterilization  as  indicated  by  negative  subcultures,  and  the 
+  sign  incomplete  sterilization. 


Antiseptic. 

Staphylococci  in  water. 

Staphylococci  in  50  per 
cent,  horse  serum. 

Phenol      

1:250- 
1:500  + 

1:50- 
1:100  + 

Salicylic  acid       

1:2,500- 
1:5,000  + 

.    1:100- 
1:250  + 

Hydrogen  perioxid 

1:3,500- 

1:1,700- 

1:8,000  + 

1:2,000  + 

lodin 

1:100,000- 

1:1,000- 

1:1,000,000  + 

1:2,500  + 

Mercuric  chlorid       .      . 

1:5,000,000- 
1:10,000,000  + 

1:25,000- 
:50,000  + 

Silver  nitrate       

1:1,000,000- 
1:10,000,000  + 

:10,000- 
:25,000  + 

Sodium  hypochlorite      .... 

1:      500,000- 
1:1,000,000  + 

:1,500- 
12,000  + 

Chloramin  T        

1:      500,000- 
1:1,000,000  + 

:2,000- 
:3,000  + 

This  decreased  efficiency  in  the  presence  of  a  protein  is  variously 
explained.  In  the  case  of  such  disinfectants  as  phenol  and  the  dye- 
stuffs,  it  is  frequently  stated  that  the  disinfectant  is  "quenched" 
or  "fixed"  by  the  protein  medium,  Adsorption  in  some  cases  may 
play  a  part,  but  in  the  case  of  salts  of  the  heavy  metals,  they  com- 
bine with  the  protein  giving  an  insoluble  non-ionizing  proteinate. 
The  low  germicidal  action  shown  by  most  antiseptics  against  pus 
is  due  in  part  no  doubt  to  the  mechanical  difficulties  of  penetrating 
the  mucoid  particles  in  the  pus. 

Young  cultures  of  bacteria  are  usually  more  resistant  than  are 
older  cultures.  This  is  especially  true  when  the  disinfectant  is 
applied  to  cultures  living  in  the  products  resulting  from  their 
metabolism.  Cultures,  the  organisms  of  which  form  spores,  become 
more  resistant  to  disinfectants  as  the  spore  stage  is  reached. 

Emulsions  as  a  rule  have  greater  germicidal  power  than  have 
solutions.  According  to  Chick  and  Martin,  emulsions  or  soapy 

8 


114  EFFECT  OF  CHEMICALS  ON  BACTERIA 

preparations  of  the  coal-tar  acids  exhibit  active  Brownian  motion. 
Often  the  bacteria  are  considerably  larger  than  the  mean  diameter 
of  the  emulsified  particles.  These  bombard  the  bacteria  and  in  this 
way  frequently  bring  them  into  intimate  contact  with  the  undiluted 
particles  of  the  disinfectant,  which  would  not  occur  in  solutions. 
Emulsions  act  upon  bacteria  first  through  physiochemical  adsorp- 
tion and  second  through  chemical  combination.  But  other  particles 
held  in  suspension  also  possess  this  power  of  adsorption,  and  hence 
the  strength  of  emulsions  is  rapidly  reduced.  Thus  the  value  of 
phenol  is  barely  impaired  by  the  presence  of  organic  matter  in  solu- 
tion, whereas  emulsified  disinfectants  are  reduced  to  one-third  or 
one-half  of  their  original  value. 

Disinfectants  of  the  Chlorin  Group.— To  this  group  belong  many 
of  the  more  active  disinfectants.  They  are  all  characterized  by  a 
chemical  instability  in  the  presence  of  organic  matter.  The  mem- 
bers of  this  group  contain  active  chlorin  in  distinction  to  inert 
chlorin,  such  as  that  in  common  salt.  The  phrase  "active  chlorin" 
does  not,  however,  necessarily  imply  that  free  chlorin  is  contained 
in  the  substance  or  liberated  by  it.  The  active  agent  may  be  hypo- 
chlorous  acid  or  some  other  compound  containing  chlorin. 

It  used  to  be  assumed  that  they  acted  mainly  by  the  liberation 
of  nascent  oxygen.  Hypochlorous  acid  decomposes  thus : 

4HC1O      =     2H2O     +     2C12     +     O2 

%  ¥\/\^  \_s 

The  chlorin  then  reacts  with  water,  liberating  more  nascent 
oxygen: 

2C12     +     2H2O      =     4HC1     +     O2 

Dakin,  however,  defines  a  substance  as  possessing  active  chlorin 
when  it  will  part  with  chlorin  either  free  or  combined  in  such  a  way 
that  it  can  effect  the  chlorination  of  bacteria  and  other  proteins. 

All  proteins  are  made  up  of  amino-acids  in  which  the  amino- 
group  of  the  one  has  reacted  with  the  carboxyl  group  of  the  other 
with  the  elimination  of  water.  This  gives  imino  NH—  groups.  It 
is  assumed  by  some  that  the  chlorin  replaces  the  hydrogen  in  this 
group,  thus: 

R 

H— C— NH2 
2C1  =  C   =  O      +    HC1 

NCI 
H— C— COOH 


DISINFECTANTS  OF  THE  CHLORIN  GROUP  115 

Or  it  may  be  that  the  extra  bonds  of  the  nitrogen  in  the  imino 
group  is  utilized : 

R 
H— c— NH2 

c  =  o 

N^Cl 
|\C1 
H— C— COOH  H— C— COOH 

I 
R 

according  to  this  explanation  hypochlorous  -acid  may  react  without 
decomposing  into  chlorin: 

R 

H— C— NH2 

I 

c  =  o 

N^H 

I  \o-ci 

H— C— COOH 
R 

In  any  case  the  chemical  and  physical  properties  of  the  protoplasm 
would  be  so  changed  as  to  be  incompatible  with  the  life  processes  of 
the  microorganisms. 

Compounds  containing  the  group— NCI— belong  to  the  class  of 
chloramins.  Their  chlorin  is  still  active  and  they  are  themselves 
active  germicides.  Such  compounds  have  been  studied  thoroughly 
by  Dakin  who  used  them  extensively  in  the  disinfecting  of  wounds 
in  the  great  European  war  of  1914-18. 

Chlorinated  lime,  or  bleaching  powder,  may  be  taken  as  a  type 
of  the  chlorin  disinfectants.  Its  precise  chemical  composition  is 
not  known  although  calcium  oxychlorid  (CaOCl2)  is  now  generally 
accepted  as  being  the  essential  agent  of  dry  bleaching  powder  and 
calcium  hypochlorite  (Ca(OCl)2)  to  be  the  active  germicide  of  the 
solution.  Although  the  reactions  which  occur  are  quite  complicated, 
it  is  certain  that  the  active  substances  are  nascent  oxygen,  chlorin, 
and  hypochlorous  acid,  and  are  probably  formed  as  follows: 

2CaOCl2  =  Ca(OCl)2  +  CaCl2 

Ca(OCl)2  +  H2COs  =  CaCOs     +     2HOC1 

2HOC1  =  2HC1  +  O2 

2HOC1  =  H2O  +  C12           +     O 

The  substance  is  extensively  used  in  the  disinfection  of  sewage, 
outhouses,  cellars,  and  for  miscellaneous  purposes.  Since  1908  it 
has  been  used  rather  extensively  in  water  purification.  In  practice 


116  EFFECT  OF  CHEMICALS  ON  BACTERIA 

from  5  to  12  or  more  pounds  of  bleaching  powder  is  used  to  each 
million  gallons  of  water.  It  cannot  be  detected  by  the  sense  of  taste 
provided  the  amount  does  not  exceed  25  pounds  to  1,000,000  gallons. 
Waters  containing  considerable  organic  material  of  any  kind  give 
rise  to  amins,  chloramins,  and  other  compounds  with  unpleasant 
flavors.  The  method,  however,  is  cheap,  reliable,  efficient,  harmless, 
and  easy  of  application. 

Formaldehyd  is  one  of  the  best  volatile  antiseptics.  If  used  in 
sufficient  concentration  and  under  proper  conditions  it  can  be 
depended  upon  for  surface  disinfection.  Although  more  penetrating 
than  sulphur  dioxid,  it  is  not  sufficient  to  depend  upon  in  deep  layers 
of  cloth  and  similar  bodies.  It  does  not  rot  nor  bleach  fabric  nor 
tarnish  metal  as  does  sulphur  dioxid.  Moreover,  formaldehyd 
unites  with  nitrogenous  substances  forming  new  chemical  compounds 
which  are  both  sterile  and  odorless.  It  is,  therefore,  good  both  as  a 
germicide  and  as  a  disinfectant. 

Although  there  are  numerous  methods  of  using,  one  of  the  best 
is  that  recommended  by  the  Pennsylvania  Department  of  Public 
Health: 

Sodium  dichromate 10    oz. 

Formalin 16    oz. 

Commercial  sulphuric  acid 1^  oz. 

The  sulphuric  acid  is  added  to  the  formalin  and  the  mixture  poured 
over  the  crystals  of  sodium  dichromate  causing  immediate  liberation 
of  formaldehyd  gas.  Five  hundred  c.c.  of  formalin  and  250  gm.  of 
sodium  dichromate  should  be  used  for  each  thousand  cubic  feet  of  air. 
The  floor  should  be  protected  against  the  heat  by  placing  the  bucket 
upon  a  brick  or  other  suitable  device. 

Sulphur  Dioxid.— Sulphur  dioxid  is  not  very  efficient  as  a  germicide; 
it  is,  however,  an  effective  insecticide.  It  is  also  good  to  use  against 
diseases  spread  by  rats,  mice,  flies,  fleas,  mosquitoes,  etc. 

Its  action  as  a  germicide  depends  upon  the  presence  of  moisture. 
The  dry  gas  is  practically  inert  against  bacteria.  It  cannot  be 
depended  upon  where  penetration  is  required,  its  action  being  merely 
upon  the  surface.  It  does  not  kill  spores.  Moreover,  it  is  a  bleaching 
agent  and  tarnishes  metals.  In  sterilization  by  means  of  sulphur, 
time  is  an  important  factor.  The  things  to  be  disinfected  should 
be  exposed  for  eight  hours  to  an  atmosphere  of  at  least  4  per  cent, 
by  volumes  of  sulphur  dioxid  gas  in  the  presence  of  water.  This 
requires  the  burning  of  4  to  5  pounds  of  sulphur  for  every  1000  cubic 
feet  of  air  space.  About  one-fifth  of  a  pound  of  water  should  be 
volatilized  for  every  pound  of  sulphur  used. 

One  method  of  using  it  follows :  The  required  quantity  of  sulphur 
is  placed  in  a  pan  which  is  put  into  a  second  larger  pan  containing 
water.  The  sulphur  is  made  into  little  craters  and  liberally  soaked 


MERCURIC  CHLORID  117 

with  alcohol.  It  is  well  to  place  the  generator  on  a  table  or  box  as 
sulphur  dioxid  is  heavier  than  air  and  hence  tends  to  sink  and  would, 
therefore,  extinguish  the  flame  if  placed  on  the  floor. 

Hydrocyanic  acid  gas  is  an  extremely  powerful  insecticide,  but 
a  poor  germicide.  It  is  used  rather  extensively  against  mosquitoes, 
lice,  bedbugs,  and  roaches,  but  on  account  of  its  highly  poisonous 
nature  it  must  be  used  with  extreme  caution.  It  is  effective  against 
bacteria  no  hardier  than  those  of  diphtheria  and  typhoid,  but  it 
cannot  be  depended  upon  as  a  general  disinfectant. 

Mercuric  chlorid  is  one  of  the  best  known  and  most  effective  of 
the  metallic  salt  disinfectants.  A  solution  of  1  to  1000  is  ample 
for  the  destruction  of  all  non-spore-bearing  bacteria,  provided  it 
comes  in  direct  contact  with  the  organisms  for  some  time.  It  is 
especially  valuable  for  disinfecting  the  hands  and  for  washing  floors, 
woodwork,  and  furniture.  It  attacks  metals  and  hence  cannot  be 
used  to  disinfect  them;  it  is  rendered  inactive  by  protein  substances; 
it  acts  on  bacteria  by  a  coagulation  of  the  protoplasm. 

Its  germicidal  value  as  usually  given*  is  too  high.  This  is  due  to 
the  fact  that  it  may  inhibit  the  growth  of  bacteria  and  in  the  planting 
of  the  cultures  the  metallic  salt  is  carried  over  into  the  new  medium, 
there  preventing  growth  but  not  necessarily  killing  the  organism. 
The  explanation  of  this  is  given  by  Miss  Chick  who  found  that  if 
bacteria  are  subjected  to  the  action  of  1:1000,  1:10,000,  or  even 
weaker  solutions  of  mercuric  chlorid,  there  is  an  interval  during 
which  some  at  least  of  them  may  be  resuscitated  by  the  timely 
administration  of  an  antidote— in  this  case  a  sulphid  solution.  If, 
however,  this  antidotal  treatment  is  not  employed,  no  amount  of 
subsequent  dilution  beyond  the  limits  of  inhibition  can  prevent  the 
death  of  the  organism. 

REFERENCES. 

Loeb:     The  Dynamics  of  Living  Matter. 
McClendon:     Physical  Chemistry  of  Vital  Phenomena. 
Dakin  and  Dunham:     Handbook  of  Antiseptics. 


CHAPTER  XIL 
INFLUENCE  OF  ARSENIC  ON  BACTERIAL  ACTIVITY. 

Occurrence  of  Arsenic. — Kunkel  showed  the  presence  of  arsenic 
in  many  rocks  and  water,  while  Czapek  states  that  traces  are  nearly 
always  present  in  soils.  Herzfeld  and  Lange  found  arsenic  in  certain 
German  raw  sugars  and  traced  it  to  the  lime  which  had  been  used 
in  the  manufacture  of  sugar.  Headden  found  some  virgin  prairie 
soils  relatively  rich  in  arsenic,  an  observation  in  accord  with  my  own 
experience.  I  have  found  arsenic  to  the  extent  of  4  parts  per  million 
in  virgin  soil;  and,  as  in  the  cases  referred  to  by  Headden,  it  did  not 
result  from  smelter  fumes  or  any  such  source,  but  was  derived  from 
the  decay  of  native  rocks.  On  the  other  hand,  Headden  found 
arsenic  in  some  cultivated  orchard  soils  to  the  extent  of  138  parts  per 
million.  He  claims  that  in  many  places  arsenic  from  spray  is  accu- 
mulating in  sufficient  quantities  to  become  injurious  to  vegetation. 
Francois,  however,  thinks  there  is  little  danger  of  the  soil's  becoming 
unfit  for  vegetation  from  the  proper  use  of  insecticides.  Grunner, 
who  found  arsenic  to  the  extent  of  from  0.026  per  cent,  to  1.426  per 
cent,  in  the  Reichenstein  soil,  is  not  so  optimistic.  An  extensive 
analysis  of  the  sprayed  orchard  soils  of  western  America  showed 
arsenic  to  be  present  in  all  of  those  soils  and  varying  from  mere 
traces  to  500  pounds  an  acre.  In  some  cases  it  occurred  to  a  depth 
of  three  or  four  feet.  The  most  interesting  fact  is  that  in  some  of 
these  soils  there  were  as  much  as  17  pounds  per  acre  of  water-soluble 
arsenic.  It  is  not,  however,  always  the  case  that  the  greatest 
quantity  of  water-soluble  arsenic  is  found  in  those  soils  which  con- 
tain the  greatest  total  quantity  of  arsenic,  for  often  soils  are  found 
which  contain  only  a  few  pounds  to  the  acre-foot,  probably  two- 
thirds  of  which  is  in  a  soluble  form.  So  the  conclusion  has  been 
reached  that  some  virgin  and  many  cultivated  soils  contain  arsenic 
in  large  quantities,  but  the  proportion  in  a  soil  is  no  index  of  the 
amount  that  is  soluble  in  water.  The  latter  is  probably  governed 
by  many  factors;  for  example,  kind  of  soil,  water-soluble  salts,  and 
form  in  which  the  arsenic  was  applied  to  the  soil. 

Factors  Influencing  Solubility.— That  the  form  in  which  the  arsenic 
is  applied  govern  largely  its  solubility  is  shown  by  an  experiment  in 
which  100  grams  of  arsenic  in  the  form  of  lead  arsenate  was  applied 
to  a  soil,  and  to  another  portion  of  the  same  soil  was  added  100 
grams  of  arsenic  in  the  form  of  Paris  green.  To  still  another  soil 


NITRIFICATION  119 

was  added  enough  arsenic  in  the  form  of  zinc  arsenite  to  make  100 
grams  of  arsenic.  These  were  carefully  mixed  and  allowed  to  stand 
for  some  time,  after  which  an  examination  was  made  for  soluble 
arsenic.  The  analysis  revealed  the  fact  that  14  per  cent,  of  the  lead 
arsenate,  30  per  cent,  of  the  zinc  arsenite,  and  over  80  per  cent,  of 
the  Paris  green  were  in  the  water-soluble  form. 

Arsenic  being  in  the  soil,  some  soluble  and  some  insoluble,  very 
naturally  suggests  the  question  as  to  what  effect  it  has  upon  the 
bacteria  of  the  soil.  Any  factor  which  influences  the  bacterial 
activities  must  indirectly  influence  the  crop  yield. 

Extensive  studies  have  been  made  on  the  influence  of  Various 
arsenic  compounds  upon  the  bacterial  flora  of  the  soil  with  the  result 
that  arsenic  was  found  to  be  a  stimulant  in  low  concentration  and 
toxic  only  in  larger  quantities.  The  extent  of  stimulation  and  toxic- 
ity  varies  greatly  with  the  specific  type  of  organism  and  the  form 
in  which  the  arsenic  is  applied. 

Ammonifiers.— Experiments  on  ammonifiers  show  that  this  class 
of  bacteria  are  not  at  first  poisoned  by  the  arsenic,  but  their  speed 
of  action  is  increased.  The  actual  results  showed  that  whereas  the 
untreated  soil  produced  in  unit  time  100  parts  of  ammonia,  soil  to 
which  60  pounds  of  arsenic  an  acre  was  applied  produced  103  parts 
of  ammonia  in  the  same  length  of  time.  And  it  was  not  until  2500 
pounds  of  arsenic  an  acre  was  applied  to  the  soil  that  the  production 
of  ammonia  was  reduced  to  one-half.  The  Paris  green,  on  the  other 
hand,  retarded  the  action  of  this  class  of  bacteria  even  in  the  lowest 
concentration  added,  and  by  the  time  600  pounds  an  acre  had  been 
applied  the  ammonia  produced  in  unit  time  had  been  reduced  to 
one-half  normal.  This  poisonous  action  of  arsenic  on  bacteria  is  in  a 
direct  relationship  to  its  solubility.  An  extremely  large  quantity 
of  lead  arsenate  would  have  to  be  applied  to  a  soil  before  it  would 
interfere  with  ammonification. 

Nitrification.  —The  nitrifying  flora  of  a  soil  are  more  resistant  and 
are  stimulated  to  a  greater  extent  by  arsenic  than  are  the  ammoni- 
fiers. Tests  made  in  soil  have  shown  that  whereas  untreated  soil 
produced  100  parts  of  nitrates  in  unit  time,  the  same  soil  to  which 
had  been  added  arsenic  in  the  form  of  lead  arsenate  at  the  rate  of 
120  pounds  an  acre  produced  178  parts  of  nitrates.  In  other  words, 
in  place  of  being  injured  by  the  arsenic,  the  bacteria  were  nearly 
twice  as  active  in  the  presence  of  this  quantity  of  arsenic  as  they 
were  in  its  total  absence.  It  was  not  until  more  than  700  pounds 
of  arsenic,  in  the  form  of  lead  arsenate,  an  acre,  had  been  applied 
to  the  soil  that  the  bacterial  activity  fell  back  to  100.  Even  when 
arsenic  in  the  form  of  lead  arsenate  was  applied  at  the  rate  of  3500 
pounds  an  acre  there  was  68  per  cent,  as  much  ammonia  produced 
as  in  the  untreated  soil.  The  Paris  green  gave  similar  results.  The 
untreated  soil  produced  100  parts  of  nitrates  in  given  time,  while 


120   INFLUENCE  OF  ARSENIC  ON  BACTERIAL  ACTIVITY 

similar  soil  to  which  arsenic  in  the  form  of  Paris  green  was  added 
produced,  under  the  same  condition,  129  parts  of  nitrates.  When, 
however,  higher  concentrations  of  arsenic  in  the  form  of  Paris 
green  were  added  it  became  toxic,  and  eventually  stopped  all 
bacterial  activity;  but  the  quantity  added  had  to  be  so  large  that 
it  is  not  likely  that  sufficient  would  ever  occur  under  agricultural 
practice. 

Arsenic,  then,  does  not  injure  the  ammonifying  or  nitrifying 
organisms  of  the  soil.  But  how  about  the  other  beneficial  bacteria 
of  the  soil?  What  effect  has  it  upon  them? 

Nitrogen  Fixation.— There  are  75,000,000  pounds  of  atmospheric 
nitrogen  resting  upon  every  acre  of  land,  but  none  of  the  higher 
plants  have  the  power  of  taking  this  directly  from  the  air.  Certain 
bacteria,  however,  can  live  in  connection  with  the  legumes  and 
assist  them  to  take  nitrogen  from  the  air.  Then  there  is  another 
set  of  nitrogen-gathering  organisms  which  live  free  in  the  soil,  and 
which  may,  under  ideal  conditions,  gather  appreciable  quantities 
of  nitrogen.  It  is  rather  possible  that  much  of  the  benefit  derived 
from  the  summer  fallowing  of  land  is  due  to  the  growth  of  this  class 
of  organisms  in  the  soil  and  storage  by  them  of  nitrogen  for  future 
generations  of  plants.  In  such  soils  they  are  both  more  active  and 
are  also  found  in  greater  numbers.  All  the  work  put  on  soil  to 
render  it  more  porous  reacts  beneficially  upon  these  organisms. 
They  not  only  require  atmospheric  nitrogen  and  oxygen,  which  are 
absolutely  essential  to  their  life  activities,  but  they  must  obtain 
them  from  within  the  soil,  for  the  minute  organisms  cannot  live 
upon  the  surface  of  the  soil  because  to  them  the  direct  rays  of  the 
sun  means  death.  How  does  arsenic  influence  this  class  of  organisms 
which  are  so  beneficial  to  the  soil,  but  which  are  so  much  more 
sensitive  to  adverse  conditions  than  are  the  other  kinds  of  bacteria? 
Arsenic  in  the  form  of  lead  arsenate,  zinc  arsenite,  and  arsenic 
trisulphid  stimulate  these  bacteria.  When  arsenic  in  the  form  of  lead 
arsenate  was  applied  to  the  soil  at  the  rate  of  500  pounds  an  acre, 
the  nitrogen-fixing  organism  gathered  twice  as  much  nitrogen  in 
unit  time  as  it  did  in  the  absence  of  arsenic.  The  Paris  green, 
however,  is  poisonous  to  this  group  of  organisms  in  the  minutest 
quantities.  This  is  most  likely  due  to  the  copper  rather  than  to  the 
arsenic  in  the  compound. 

How  Does  the  Arsenic  Act?— It  may,  therefore,  be  concluded  that 
arsenic  stimulates  all  the  beneficial  bacteria.  But  how  does  it  act? 
Will  it  stimulate  for  a  short  time  and  then  allow  the  organism 
to  drop  back  to  its  original  or  to  a  lower  level  as  does  alcohol  and 
various  stimulants  when  given  to  animals?  Will  it  act  as  does 
caffeine— continue  to  stimulate?  From  the  results  on  men  and 
horses  the  former  might  be  expected,  for  although  the  arsenic  eaters 
of  India  and  Hungary  maintain  that  the  eating  of  arsenic  increases 


tiOW  DOES  THE  ARSMIC  ACT  121 

their  endurance,  and  there  is  considerable  evidence  to  indicate  this, 
it  is  only  for  a  short  time.  If  the  use  be  discontinued  the  arsenic 
eaters  cannot  endure  as  much  physical  exertion  as  can  others  who 
are  not  addicted  to  the  drug.  Many  European  horse  dealers  place 
small  quantities  of  arsenic  in  the  daily  corn  given  to  the  horse,  for 
they  find  it  improves  the  coat  of  the  horse.  If  a  horse,  however,  has 
been  doped  on  arsenic  for  a  long  time  it  seems  necessary  to  continue 
the  practice;  otherwise,  the  animal  rapidly  "loses  his  condition." 

Similar  results  might  be  expected  with  the  bacteria,  and  experi- 
ments have  shown  that  although  during  the  first  few  weeks  the 
bacterial  activity  of  soils  containing  small  quantities  of  arsenic  is 
much  greater  than  it  is  in  a  similar  soil  without  arsenic,  this  activity 
continues  to  get  less  and  less,  until  at  the  end  of  several  weeks  it  is 
no  greater  than  in  soil  containing  no  arsenic.  It  is  interesting  to 
note  that  if  proper  aeration  is  maintained  bacterial  activity  never 
becomes  lower  than  in  untreated  soil. 

Now  why  this  stimulating  influence  of  arsenic  upon  soil  bacteria?  A 
similar  condition  has  been  found  to  exist  when  soils  are  treated  with 
carbon  bisulphid,  chloroform,  or  other  disinfectants,  or  even  when 
the  soil  is  heated.  Many  theories  have  been  offered  to  account  for 
it,  but  probably  the  most  interesting  is  the  one  held  by  Russell  and 
Hutchinson.  They  maintain  that  within  the  soil  are  microscopic 
plants,  bacteria,  and  also  microscopic  animals,  protozoa.  The 
minute  animals  are  continually  feeding  upon  the  minute  plants, 
with  the  result  that  the  bacterial  plants  cannot  multiply  as  they 
could  in  the  absence  of  the  protozoa.  Now  when  a  weak  solution 
of  an  antiseptic  is  applied  to  the  soil  it  kills  many  of  the  protozoa, 
and  the  bacteria  being  no  longer  preyed  upon  by  their  natural  foe 
rapidly  multiply.  As  the  antiseptic  evaporates  the  few  remaining 
protozoa  start  to  multiply  and  soon  are  able  to  keep  in  check  the 
bacterial  flora  of  the  soil.  So  within  the  soil  one  species  preys  upon 
another.  It  is  possible  that  microscopic  forms  of  life  wage  within 
the  soil  battles  as  terrific  as  those  waged  by  the  higher  forms  of  life 
upon  the  earth's  surface. 

It  is  likely  that  this  is  one  of  the  ways  in  which  arsenic  stimulates- 
the  bacterial  activities  of  the  soil.  It  acts  more  readily  upon  the 
protozoa  than  upon  the  bacteria.  After  the  arsenic  has  been  in  the 
soil  for  some  time  it  may  become  insoluble  or  some  of  it  may  be 
changed  by  molds  into  a  gas  arsine  and  pass  into  the  air.  Then  the 
few  protozoa  which  have  not  been  destroyed  by  its  presence  rapidly 
multiply  and  soon  hold  the  bacteria  in  check. 

This,  however,  is  not  the  only  way  in  which  arsenic  acts,  for  pure 
cultures  of  the  Azotobacter  have  been  obtained  from  these  soils,  and 
it  is  found  that  these  are  so  stimulated  that  they  bring  about  greater 
changes  in  the  presence  of  arsenic  than  they  do  in  its  absence.  This 
is  due  to  the  action  of  the  arsenic  upon  these  minute  specks  of  living 


122    INFLUENCE  OF  ARSENIC  ON  BACTERIAL  ACTIVITY 

protoplasm,  causing  them  to  utilize  their  food  more  economically 
in  the  presence  of  arsenic  than  in  its  absence.  This  is  similar  to  the 
influence  of  the  arsenic  upon  the  cells  within  the  horse. 

Other  experiments  have  demonstrated  that  the  addition  of  arsenic 
to  a  soil  increases  the  liberation  of  the  insoluble  plant-foods  of  the 
soil,  especially  of  the  phosphorus.  Thus  arsenic  by  various  means 
stimulates  all  the  bacterial  activities  of  the  soil,  and  these  increased 
activities,  as  experiments  have  shown,  are  reflected  in  greater  crops. 
This  increased  growth  must  be  looked  upon  as  due  to  a  stimulus 
and  not  to  the  direct  nutritive  value  of  the  substance  added.  Soils 
so  treated  would  produce  larger  crops  and  wear  out  more  quickly 
than  would  untreated  soils.  It  is  interesting  and  important  to  know 
that  arsenic  has  to  be  applied  to  a  soil  in  enormous  quantities  before 
it  retards  microscopic  plant  life,  and  probably  before  it  retards  the 
growth  of  higher  plants. 

The  data  available  prove  conclusively  that  the  arsenical  com- 
pounds, with  the  single  exception  of  Paris  green,  stimulate  the 
nitrogen-fixing  organisms  of  the  soil  and  that  this  influence  varies 
qualitatively  but  not  quantitatively  with  the  various  soils.  The 
results  also  bring  out  the  fact  that  both  the  anion  and  the  cation  of 
the  compounds  have  a  marked  influence  upon  the  growth  of  the 
organisms.  With  some  compounds  both  the  anion  and  cation  act 
as  stimulants,  but  with  other  compounds  one  stimulates  and  the 
other  retards.  It  is  likely  that  little  or  no  influence  is  exerted  upon 
the  nitrogen-gathering  organisms  by  the  sodium  of  sodium  arsenate 
and  that  the  stimulating  influence  noted  with  dilute  solutions  and 
the  toxic  influence  exerted  with  more  concentrated  solutions  are  due 
entirely  to  the  arsenic.  It  is  rather  likely  that  the  stimulating 
influence  which  Riviere  and  Bouilhac  have  found  sodium  arsenate 
to  have  upon  wheat  and  oats  is  an  indirect  effect  which  is  exerted 
upon  the  bacterial  flora  of  the  soil  and  which  in  turn  influences  the 
yield  of  the  various  grains. 

Both  the  anion  and  cation  undoubtedly  act  as  stimulants  in  the 
lead  arsenate.  Stoklasa  has  shown  that  lead  when  present  in  soil 
stimulates  the  growth  of  higher  plants.  This  he  ascribes  to  the 
catalytic  action  of  these  elements  on  the  chlorophyll.  The  results 
reported  indicate  that  it  is  due  to  the  influence  of  the  compounds 
upon  the  biological  transformation  of  the  nitrogen  in  the  soil.  The 
fact  that  the  lead  plays  no  small  part  in  the  stimulating  influence 
is  borne  out  by  the  work  of  Lipman  and  Burgess  who  found  lead  to 
stimulate  nitrifying  organisms. 

Paris  green  is  toxic  to  the  nitrogen-fixing  organism  in  the  lowest 
concentration  tested.  This  is  due  to  the  copper  and  not  to  the 
arsenic,  as  it  is  well  known  that  the  copper  ion  is  a  strong  poison 
to  many  of  the  lower  plants.  Brenchley  found  it  to  be  toxic  to 
higher  plants  when  present  in  water  to  the  extent  of  one  part  in 


HOW  DOES  THE  ARSENIC  ACT  123 

4,000,000,000.  Although  Russell  states  that  it  is  not  as  toxic  in 
soil  as  in  water,  Darbishire  and  Russell  found  it  to  be  toxic  in  soils, 
and  they  failed  to  get  a  stimulating  influence  with  it.  Monte- 
matini  has  noted  a  stimulation  with  copper  sulphate  when  used  in 
dilute  solutions.  This,  however,  may  have  been  due  to  the  anipn 
and  not  to  the  cation,  as  sulphates  do  stimulate  plants  by  their  action 
on  insoluble  constituents  of  the  soil.  The  same  interpretation  could 
be  placed  upon  the  results  obtained  by  Lipman  and  Wilson  and  also 
those  reported  by  Voelcker  in  which  they  noted  a  stimulation  with 
copper  salts.  Clark  and  Gage  have  found  that  very  dilute  solutions 
of  copper  have  an  invigorating  influence  upon  bacterial  activity.  In 
order  that  the  stimulation  may  be  noted  the  copper  must  be  present 
in  small  quantities.  Jackson  found  that  1  part  of  copper  sulphate 
in  50,000  parts  of  water  kill  Bacillus  coli  and  Bacillus  typhosus. 
Kellermann  and  Beckwith  found  that  the  common  saprophytic 
bacteria  are  more  resistant  to  copper  than  is  B.  coli.  There  is  con- 
siderable evidence  that  copper  stimulates  the  ammonifying  and 
nitrifying  organisms  of  the  soil,  but  these  results  show  the  nitrogen- 
fixing  organisms  of  the  soil  to  be  very  sensitive  to  copper,  and  if  it 
is  to  act  as  a  stimulant  it  must  be  in  extremely  dilute  solutions.  The 
toxicity  of  the  copper  in  the  Paris  green  is  great  enough  in  the 
dilution  of  10  parts  in  1,000,000  to  offset  the  great  stimulating 
influence  of  the  arsenic  in  combination  with  it. 

The  marked  stimulating  influence  noted  where  the  arsenic  trisul- 
phid  is  used  is  very  probably  due  to  the  stimulating  action  of  both 
the  arsenic  and  sulphur.  Demolon  attributed  much  of  the  fertilizing 
action  of  sulphur  to  its  action  upon  bacteria,  and  Vogel  found  that 
sulphur  decidedly  increased  the  activity  of  the  nitrogen-fixing  organ- 
isms. The  results  which  Russell  and  Hutchinson  obtained  with 
calcium  sulphid  are  interesting  in  this  connection.  They  found  that 
after  thirty  days  there  were  five  times  as  many  organisms  in  a  soil 
to  which  calcium  sulphid  had  been  added  as  in  an  untreated  soil,  and 
the  yield  of  ammonia  and  nitrates  in  the  same  length  of  time  was 
one-third  greater  in  the  treated  soil  than  in  the  untreated  soil.  This 
in  turn  reacts  upon  the  crop  harvested,  as  shown  by  Shedd. 

The  first  part  of  the  curve  for  zinc  arsenite  nearly  coincides  with 
that  of  sodium  arsenate,  save  that  zinc  arsenite  stimulates  in  greater 
concentrations  than  does  sodium  arsenate.  This  is  partly  due  to 
the  difference  in  solubility  of  the  two  compounds,  but  there  is 
another  factor— that  the  zinc  also  acts  as  a  stimulant.  Latham 
found  that  small  quantities  of  zinc  stimulated  algse.  The  same 
results  have  been  obtained  by  Silberberg  in  working  with  higher 
plants.  Ehrenberg  concludes  that  zinc  salts  are  always  toxic  when 
the  action  is  simply  on  the  plant,  but  that  they  may  lead  to  increased 
growth  through  some  indirect  action  on  the  soil.  He  found  that  zinc 
stimulated  plant  growth  in  soils,  but  when  the  soil  was  sterilized  the 


124    INFLUENCE  OF  ARSENIC  ON  BACTERIAL  ACTIVITY 

zinc  became  toxic.  Lipman  and  Burgess  have  shown  that  it  stimu- 
lates the  nitrifying  organisms  and  that  the  influence  is  shown  in  the 
crop  yield.  The  great  variation  in  the  results  reported  by  the  differ- 
ent investigators  for  zinc,  arsenic,  and  lead  is  probably  due  to  the 
fact  that  it  modifies  the  bacterial  flora  of  the  soil.  When  heated 
soil  or  water  cultures  are  used  a  different  result  is  noted.  This, 
however,  is  not  the  only  factor,  for  these  results  show  a  marked 
difference  in  soil  and  in  water  culture.  The  lead  arsenate  stimulates 
the  nitrogen-fixing  organisms  when  placed  in  soils  but  becomes 
highly  toxic  to  the  same  organisms  when  placed  in  nutritive  solutions. 
The  difference  is  due  in  part  to  adsorption  by  the  soil,  but  this 
would  have  to  be  attributed  to  the  silica  compounds  of  the  soil,  for 
the  nitrogen-fixing  organisms  are  stimulated  by  arsenic  in  quartz 
sand  free  from  organic  colloids.  In  this  case  the  arsenic  becomes 


FIG.   18. — Graph  showing  the  effect  of  aeration  on  the  nitrogen-fixing  activity  of  soil- 
containing  compounds  of  arsenic.     (Soil  Science.) 


concentrated  at  the  surface,  layers  of  the  silica  leaving  the  inner 
part  of  the  water  film  comparatively  free  from  arsenic,  in  which 
the  microorganisms  multiply  and  carry  on  their  metabolic  processes. 
This  being  the  case,  it  is  probable  that  a  water  solution  weak  enough 
to  stimulate  bacteria  could  be  found.  A  great  difference,  however, 
between  the  solution  and  the  sand-culture  method  is  the  greater 
aeration  in  the  latter  than  in  the  former.  That  the  aeration  of  a 
cultural  medium  does  play  a  great  part  in  determining  the  activity 
of  the  nitrogen-fixing  powers  of  a  soil  is  strikingly  brought  out  in 
Fig.  18. 

It  is  remarkable  how  the  aeration  of  the  soil  or  the  filtering  of  the 
soil  extract  can  prevent  the  high  loss  of  nitrogen  which  is  noted  at 
first  in  the  unaerated  soil.  This  cannot  be  attributed  directly  to  the 
denitrifying  organisms;  otherwise,  it  would  not  be  removed  by  filtra- 


HOW  DOES  THE  ARSENIC  ACT 


125 


tion.  The  graphs  also  bring  out  the  fact  that  adding  arsenic  and 
filtering  the  soil  only  shift  for  the  time  the  equilibrium  within  the 
soil,  which  later  tends  to  regain  its  old  equilibrium.  This  is  a  condi- 
tion that  coincides  well  with  what  would  be  expected  if  the  limiting 
element  were  some  other  microscopic  forms  of  life.  The  filter 
would  not  separate  them  quantitatively,  and  it  is  possible  that  the 
arsenic  has  only  a  selective  influence.  Later,  many  of  the  organ- 
isms become  accustomed  to  its  presence;  or,  what  is  more  likely,  the 
arsenic  becomes  fixed  within  the  soil. 

That  this  limiting  factor  is  a  thermolabile  body  is  brought  out 
more  clearly  by  Fig.  19.  The  quantity  of  nitrogen  fixed  by  the 
unheated  soil  receiving  no  arsenic  has  been  taken  as  100,  the  heated 
soil  with  and  without  arsenic  being  compared  with  this. 


320^ 


300  ;i 


FIG.  19. — Graph  showing  the  effect  of  the  heat  on  the  nitrogen-fixing  power  of  soil 
treated  and  not  treated  with  arsenic. 

The  heating  of  the  soil  extract  to  50°  C.  for  fifteen  minutes  has 
exactly  the  same  influence  measured  in  terms  of  nitrogen  fixed  as 
does  0.0728  gm.  of  lead  arsenate.  The  stimulating  influence  of  heat 
is  noted  even  in  the  presence  of  arsenic  and  reaches  its  maximum 
effect  in  the  absence  of  arsenic  at  60°,  and  in  the  presence  of  arsenic 
at  65°  C.  Above  these  temperatures  there  is  a  decline  in  the  nitrogen 
fixed.  Even  soils  inoculated  with  solutions  which  had  been  heated 
to  a  temperature  of  85°  fixed  nitrogen;  at  least  there  is  more  nitrogen 
accumulated  in  such  soil  than  in  that  inoculated  with  the  untreated 
soil  solution.  The  results  indicate  that  many  of  the  organisms  which 
take  part  in  the  gathering  of  nitrogen  in  soils  are  very  resistant  to 
heat.  It  is  also  significant  that  the  greatest  stimulating  influence 


126    INFLUENCE  OF  ARSENIC  ON  BACTERIAL  ACTIVITY 

is  exerted  in  soil  which  had  been  inoculated  with  solutions  heated 
just  above  that  point  which  Cunningham  and  Lohnis  found  to  be 
the  thermal  death  point  of  soil  protozoa. . 

REFERENCES. 

Greaves,  J.  E.:  The  Occurrence  of  Arsenic  in  Soils  (Bichem.  Bull.,  1913,  ii,  519- 
523). 

Greaves,  J.  E. :  Some  Factors  Influencing  Ammonification  and  Nitrification  in 
Soil  (Centr.  f.  Bakt.,  Bd.  xxxix,  Abt.  II,  1913,  542-560). 

Greaves,  J  E.:  Stimulating  Influence  of  Arsenic  upon  the  Nitrogen-fixing  Organ- 
isms of  the  Soil  (Journal  Agricultural  Research,  1916,  vi,  389-416). 


CHAPTER  XIII. 

EFFECT  OF  HEAT  AND  VOLATILE  ANTISEPTICS  ON 
SOIL  BACTERIA. 

SOILS  are  often  heated,  steamed,  or  treated  with  volatile  or  non- 
volatile antiseptics  both  for  experimental  and  practical  purposes. 
The  process  is  not  sufficient  to  destroy  all  forms  of  life  within  the  soil. 
It  only  destroys  some  of  the  weaker  species  and  the  aim  is  usually  to 
destroy  an  injurious  species.  Yet  the  process  is  often  referred  to 
as  sterilization.  In  view  of  the  fact  that  they  fail  to  render  the  soil 
sterile,  some  workers  prefer  the  terms  partial  sterilization  or  pas- 
teurization, which  more  accurately  describe  the  process. 

Although  it  was  well  known  that  the  kiln-burning  of  clay  produced 
a  far-reaching  chemical  and  physical  effect,  yet  soil  investigators 
considered  that  the  process  of  sterilization  produced  no  change 
either  in  the  mechanical  nature  or  chemical  composition  of  a  soil 
until  the  work  of  Frank  appeared  in  1888.  He  found  that  heated 
soils  contained  a  great  deal  more  soluble  matter  than  unheated  soil, 
peaty  soils  containing  more  than  twice  as  much  and  heated  sandy 
soils  not  quite  twice  as  much.  This  increased  soluble  matter  he 
considered  sufficient  to  account  for  the  increase  in  crops  which  was 
often  found  to  follow  the  heating  of  a  soil. 

A  great  impetus  was  given  to  the  work  in  1894  by  Oberlin  in 
Germany  and  Girard  in  France  who  found  that  the  application  of 
carbon  bisulphid  increased  the  crop-producing  power  of  the  soil. 
Oberlin  found  that  vineyards  treated  with  carbon  bisulphid  to  kill 
phylloxera  showed  greatly  increased  productivity  after  the  treat- 
ment, and  he  founded  on  this  his  system  of  grape  culture,  where 
fallowing  and  rotation  could  be  dispensed  with  in  the  resetting  of 
vineyards.  Girard  noticed  that  soil  treated  with  carbon  bisulphid 
for  the  purpose  of  combating  a  parasitic  disease  of  sugar-beet  was 
more  productive  than  it  was  before  such  treatment.  The  beneficial 
influence  of  the  treatment  extended  even  into  the  second  year. 
These  facts  stimulated  investigation  and  created  much  discussion, 
particularly  as  to  the  manner  of  its  action.  No  working  hypothesis 
was,  however,  formulated  until  1899  when  Koch  announced  his 
direct  "stimulation  theory,"  since  which  time  numerous  theories 
have  been  formulated  to  account  for  the  noted  phenomena. 

Influence  on  Plant.— The  use  of  carbon  bisulphid  at  the  rate  of  2904 
pounds  an  acre  resulted  in  a  gain  of  15  to  46  per  cent,  in  the  yield 


128  EFFECT  OF  HEAT  ON' SOIL  BACTERIA 

of  wheat  grain  and  of  21  to  80  per  cent,  in  wheat  straw.  The  yield 
of  potatoes  was  similarly  increased  by  5  to  38  per  cent,  and  that  of 
beets  from  18  to  29  per  cent.  Although  the  yields  of  the  legumes 
were  not  always  increased,  yet  some  fields  of  clover  treated  with 
carbon  bisulphid  gave  increases  of  119  per  cent. 

Wollny  clearly  showed  that  the  application  of  carbon  bisulphid  to 
a  soil  within  the  growing  season  may  lead,  according  to  the  amount 
introduced,  to  a  complete  destruction  of  the  growing  crop,  or  to 
a  temporary  retardation  merely,  involving  a  greater  or  slighter 
depression  in  the  production  of  plant  substance.  Its  application 
several  months  before  planting  increases  the  fertility  of  the  soil 
to  a  considerable  extent.  This  influence  is  felt,  according  to  the 
amount  of  carbon  bisulphid  used,  through  one  or  several  growing 
seasons,  after  which  if  no  manure  or  fertilizer  has  been  applied  a 
marked  decrease  in  the  yields  becomes  evident. 

There  was  the  dark  green  color  and  the  vigorous  development  of 
the  plants  together  with  the  decided  tendency  of  grain  crops  to 
lodge  just  as  if  too  great  quantities  of  nitrogen  were  at  their  disposal. 
These  facts  led  Heinze  to  conclude  that  on  the  whole  we  must  seek 
the  cause  of  the  beneficial  effect  of  carbon  bisulphid  on  the  soil  in 
the  enormous  increase  of  soil  organisms  at  the  proper  time,  thus 
rendering  available,  or  possibly  increasing,  the  nitrogen  supply  to 
growing  plant. 

The  large  amounts  of  nitrogen  thus  made  available  to  the  crops 
are  derived  partly  from  the  soil  and  partly  from  the  atmosphere. 
Kruger  and  Heinze  not  only  demonstrated  that  soils  treated  with 
carbon  bisulphid  showed  an  increase  in  their  total  nitrogen  content, 
but  also  that  the  increase  was  the  result  of  the  more  vigorous  growth 
of  the  nitrogen-fixing  Azotobacter  species.  This,  Heinze  considers, 
resulted  from  the  initial  suppression  of  amid-ammonia  formation 
and  nitrification  which  would  create  favorable  conditions  for  the 
development  of  nitrogen-fixing  flora.  Later  there  would  be  more 
intense  transformation  of  the  bacterial  proteins  and  of  other  nitrog- 
enous organic  substances  into  amino-  and  ammonia  compounds 
which  would  result  in  a  more  vigorous  nitrification,  thus  placing 
at  the  disposal  of  the  plant  an  abundant  and  uniform  supply  of 
soluble  nitrogen  compounds.  The  various  organic  materials  in  the 
soil— such  as  plant  residues,  pectins,  pentosans,  humic  substances, 
and  the  like,  together  with  the  rapid  growth  of  algre  and  molds— 
may  furnish  the  carbon  food  for  the  Azotobacter  species. 

Effect  on  Properties  of  Soil.— Egorow,  who  investigated  the  effects  of 
carbon  bisulphid  upon  the  physical  properties  of  the  soil,  found  that 
(1)  the  capillary  rise  of  water  in  the  soil  treated  with  carbon  bisul- 
phid to  be  slower  than  in  the  untreated;  (2)  the  moisture  content 
is  reduced  considerably,  especially  in  peaty  soils;  and  (3)  the  water- 
holding  capacity  of  the  soil  is  decreased*  Thus,  he  concludes  that 


EFFECT  ON  PROPERTIES  OF  SOIL  129 

the  treatment  of  soils  with  carbon  bisulphid  acts  unfavorably  upon 
the  water  content  of  the  soil. 

Other  characteristic  effects  of  treatment  with  volatile  antiseptics 
reported  by  various  investigators  are : 

1.  An  initial  decrease  in  the  number  of  bacteria  followed  by  a 
long-continued  increase.    A  careful  piece  of  experimentation  illus- 
trating this  is  that  of  Fred  who  used  loam  soil  (mixed  with  sand) 
and  found  that  2  per  cent,  carbon  bisulphid  has  little  effect  upon 
the  moisture  content  of  the  soil.    With  varying  percentages  of  ether 
(together  with  2  per  cent,  of  sugar)  in  the  soil,  he  finds  an  initial 
depression  in  bacterial  numbers  followed  by  a  considerable  increase 
in  eight  hours,  4  per  cent,  giving  the  maximum  count. 

2.  A  disturbance  of  the  equilibrium  of  the  bacteria,  by  which 
certain  types  multiply  more  rapidly  than  others.     Hiltner  and 
Stormer  found  that  under  normal  conditions  there  is  a  certain 
equilibrium  established  among  the  various  groups  of  soil  bacteria, 
and  that  the  organisms  capable  of  growing  on  meat  extract  gelatin 
are  composed  of  Streptothrir  species  20  per  cent.,  gelatin-liquefying 
species  75  per  cent.,  and  the  non-liquefying  species  5  per  cent. 

When  carbon  bisulphid  is  applied  to  a  soil,  its  bacterial  inhabitants 
are  injured,  though  not  completely  destroyed,  the  injury  varying 
with  the  changing  conditions  of  temperature,  moisture,  and  amount 
of  carbon  bisulphid  applied,  as  well  as  with  the  duration  of  its  action. 
Not  all  of  the  bacterial  species  are  depressed  in  their  development 
to  an  equal  extent,  the  injury  being  most  pronounced  in  the  strepto- 
thrix  species  and  least  pronounced  in  the  gelatin-liquefying  species. 
The  depressing  action  of  carbon  bisulphid  disappears  after  a  shorter 
or  longer  interval  and  is  followed  by  a  rapid  multiplication  of  the 
microorganisms  in  the  soil.  The  equilibrium  having  been  destroyed, 
however,  the  new  development  follows  along  different  channels, 
and  there  occurs  not  only  an  enormous  increase  in  the  total  number 
of  soil  bacteria,  but  also  an  abnormal  predominance  of  certain 
species.  The  new  conditions  thus  established  for  a  time  favor  a 
more  ready  utilization  of  the  stores  of  soil  nitrogen,  and  likewise  the 
fixation  of  atmospheric  nitrogen  by  certain  bacterial  species.  These 
conclusions  are  borne  out  by  the  work  of  Lipinan  and  Brown  who 
examined  abnormal  soil  after  applying  carbon  bisulphid  in  various 
quantities  alone,  and  in  combination  with  muriate  of  potash  and 
acid  phosphate.  They  then  determined  the  ammonifying,  nitrifying, 
denitrifying,  and  nitrogen-fixing  powers  of  the  soil.  They  concluded 
that  in  normal  soil  flora  the  different  groups  occur  in  fairly  definite 
relations  which  are  evidently  disturbed  by  the  addition  of  carbon 
bisulphid,  which,  destroying  the  bacterial  equilibrium  prepares  the 
way  for  an  entirely  new  bacterial  development  whereby  certain 
species  become  far  more  prominent  than  previously.  This  applies 
especially  to  the  nitrifying  and  nitrogen-fixing  bacteria. 
9 


130 


EFFECT  OF  HEAT  ON  SOIL  BACTERIA 


3.  A  slight  initial  increase  in  ammonia  content,  followed  by  a 
considerable  increase  in  the  production  of  ammonia.  This,  although 
noted  by  the  majority  of  workers,  is  especially  brought  out  by  the 
work  of  Russell  and  Hutchinson,  as  is  illustrated  by  the  following 
results  from  their  work: 


Nitrogen  pres- 
ent as  NHi 
(p.p.m.). 

Nitrogen  present 
as  NO»  (p.p.m.) 

Total  nitrogen  present  as 
NH,  and  NO,. 

At 
begin- 
ning. 

After 
23  days. 

At  begin- 
ning. 

After 
23  days. 

At  begin- 
ning. 

After 
23  days. 

Gain  in 
23  days. 

Untreated  soil     .... 
SoH  heated  2  hours  at  98°  C. 
Soil    treated    with    toluene 
which  was  then  evapor- 
ated       

1.8 
6.5 

5.0 

7.2 

1.7 
43.8 

27.8 
14.5 

12.0 
13.0 

12.0 
11.0 

16.0 
12.0 

12.0 
10.0 

13.8 
19.5 

17.0 
18.2 

17.7 
55.8 

39.8 
45.5 

3.9 
36.3 

22.8 
6.3 

Soil    treated    with    toluene 
which  was  not  removed  . 

4.  A  depression  of  the  processes  by  which  ammonia  is  converted 
into  nitric  acid,  and  a  very  slow  recovery  of  the  activity  of  the  bac- 
teria concerned,  as  a  result  of  which  ammonia  accumulates  in  the 
soil.    Warington,  in  his  early  investigation  on  the  biological  nature 
of  nitrification,  observed  that  when  air  containing  carbon  bisulphid 
was  passed  through  the  soil  the  process  was  inhibited,  whereas  C. 
de  Briailles  noted  that  during  the  winter  the  carbon  bisulphid  seemed 
to  exert  a  harmful  influence  on  the  accumulation  of  nitrates.    How- 
ever, with  the  first  open  weather  in  spring  the  reverse  seemed  to  be 
true— the  carbon  bisulphid  caused  a  marked  increase  in  nitrates  over 
the  untreated. 

5.  An  increase  in  the  rate  at  which  oxidation  takes  place  in  the 
soil.    In  a  study  of  oxidation  in  soils  and  its  relationship  to  produc- 
tiveness,  Darbishire  and   Russell  found  that  the  absorption   of 
oxygen  by  soil  is  mainly  brought  about  by  the  action  of  micro- 
organisms and  is  greatly  diminished  if  the  soil  has  been  previously 
heated  to  120°  C.    When  heated  to  95°  C.,  it  was  found  that  the 
rate  of  oxidation  on  a  sand,  two  loams,  and  a  chalky  soil,  instead  of 
being  reduced  was  considerably  increased,  as  was  the  case  after 
treatment  with  and  removal  of  volatile  antiseptics,  such  as  toluene, 
chloroform,  carbon  bisulphid,  and  other  volatile  antiseptics. 

The  rates  of  oxidation  of  heated  soils  were  as  follows : 


Milligrams  of  oxygen  absorbed  in 


Hop  garden  soil,  unheated     . 
Hop  garden  soil,  heated  to  95°  C. 
Garden  soil,  unheated 
Garden  soil,  heated  to  95°  C. 


3  days. 
3.7 
6.0 
7.5 

16.9 


6  days. 

5.2 

8.2 
10.2 
27.2 


9  days. 

7.0 
12.0 
15.5 
33.2 


EFFECT  OF  PROPERTIES  OF  SOIL  131 

6.  It  has  been  repeatedly  demonstrated  by  many  workers  that 
both  heat  and  antiseptics  destroy  all  or  part  of  the  protozoa  found 
in  the  soil,  depending  on  the  degree  of  heat  applied  or  the  strength 
of  antiseptic  used. 

7.  Some  workers  have  found  antiseptics  and  heat  to  depress 
denitrification  in  soil.    Both  Wagner  and  Morgan  found  that  carbon 
bisulphid  kills  denitrifying  organisms. 

8.  Especially  significant  is  the  fact  that  there  is  a  considerable 
increase  in  the  soluble  matter  in  the  heated  soil,  not  only  of  inorganic 
matter,  as  phosphorus  and  potash,  but  even  more  in  the  organic 
matter  made  soluble.    Stoklasa  holds  that  the  plants  are  able  to  get 
more  phosphate-ions  from  a  soil  as  a  result  of  the  disintegration  of 
the  bacteria  killed  by  the  treatment  with  carbon  bisulphid. 

Fred  found  that  the  application  of  carbon  bisulphid  to  a  soil 
increases  the  insoluble  compounds  of  nitrogen  and  sulphur  as  well  as 
the  bacterial  activities.  Lyon  and  Bizzell  determined  the  effect  of 
sterilizing  soils  by  steam  on  the  water-soluble  material  and  found 
that  steaming  the  soil  at  two  atmospheres  reduced  the  nitrates  to 
nitrites  and  ammonia,  but  that  most  of  the  ammonia  is  formed  from 
organic  nitrogen  in  the  soil . 

9.  Although  the  majority  of  workers  report  an  increase  in  nitrogen 
fixed  in  a  soil  treated  with  carbon  bisulphid,  yet  Koch  reports  cases 
in  which  carbon  bisulphid  added  to  a  soil  containing  fairly  large 
quantities  of  cane  sugar  has  resulted  in  a  weakening  rather  than  in 
a  strengthening  of  their  nitrogen-fixing  powers.     The  increase  in 
nitrogen  fixation  may  at  times  be  very  pronounced,  as  may  be 
seen  from  the  following  experiments  in  which  tumblers  containing 
soil  were  all  carefully  sterilized  and  half  of  them  placed  in  the 
.incubator  in  the  sterile  condition.    To  the  others  was  added  a  soil 
extract  prepared  by  shaking  one  part  of  soil  with  two  parts  of  sterile 
distilled  water  for  three  minutes.     After  standing  for  about  five 
minutes  the  liquid  was  decanted  and  10  c.c.  portions  were  used  to 
inoculate  the  soil.    Before  inoculating,  this  extract  was  placed  in 
thin-walled  test-tubes  in  10  c.c.  portions  and   then   kept   at  the 
required  temperature  for  exactly  fifteen  minutes  before  adding  to 
the  soil.    The  moisture  content  was  made  up  to  18  per  cent,  and 
the  whole  incubated  for  twenty  days.    The  milligrams  of  nitrogen 
fixed  under  the  varying  treatments  were  as  follows: 

Milligrams 
Temperature  of  soil  extract  (°  C.).  nitrogen-fixed. 

Room 5.11 

50 9.00 

55 14.14 

60 16.38 

65 14.42 

70 13.02 

75 11.34 

80 12.66 

85 .      .      10.36 


132  EFFECT  OF  HEAT  ON  SOIL  BACTERIA 

10.  Although  strongly  contested  by  many  experimenters,  there 
are  some  workers  who  have  produced  strong  evidence  that  the  heat- 
ing of  a  soil  destroys  toxins  which  have  been  formed  in  it  due  either 
to  bacterial  activity  or  to  plant  growth.  Fletcher  grew  plants  in 
a  nutrient  solution  until  he  considered  it  to  be  made  toxic  by  plant 
excreta.  He  then  steamed  it  at  a  pressure  of  150  pounds  for  two 
hours.  Flakes  of  an  organic  substance  were  thrown  down,  which 
did  not  dissolve  on  removal  of  pressure.  This  substance  he  con- 
sidered to  be  the  toxic  excreta. 

The  principal  changes  produced  in  and  by  sterilizing  soil  are  thus 
summarized  by  Johnson : 
I.  Destruction  of  Life: 

(a)  Normal  soil  flora  and  fauna,  desirable  and  undesir- 
able forms  of  bacteria,  fungi,  protozoa,  and  higher 
animals. 
(6)  Plant  parasites,  especially  pathogenic  bacteria,  fungi, 

nematodes,  and  injurious  soil-infection  insects, 
(c)  Propagative  organs  of  higher  plants,  especially  weed 

seeds. 

II.  Immediate  Chemical  Action  (formation  of  toxic  and  bene- 
ficial compounds) : 

(a)  Decomposition  of  organic  material  resulting  in  the 
formation  of  ammonia,  carbon  dioxid,  and  various 
new  and  complex  organic  compounds. 
(6)  Decomposition  of  inorganic  material,  reduction  of 
nitrates  and  nitrites  to  ammonia  and  increased  solu- 
bility of  potassium,  phosphorus,  and  other  salts. 

III.  Biochemical  Action : 

(a)  Increased  ammonification,  particularly,  and  modified 
nitrification,  denitrification,  and  nitrogen-fixation. 

IV.  Physical  Action : 

(a)  Absorption  capacity  of  soil  modified  for  water,  gases 

and  salts. 
(6)  Increased  concentration  of  soil  solution. 

(c)  Modified  capillarity,  colloidal  state,  and  mechanical 

condition. 

(d)  Modified  color  and  odor. 

V.  Action  on  Organisms  Growing  in  Sterilized  Soils: 
(a)  Lower  organisms. 

1.  Increased  development  due  to  reduced  competi- 

tion, increased  food  supply,  destruction  of 
"bacterio-toxins,"  "stimulation,"  byproducts 
added  or  formed,  or  other  causes. 

2.  Retardation  in  growth  in  rare  cases  due  to  inju- 

rious conditions  produced. 


HILTNER  AND  STORMER'S  "INDIRECT"   THEORY        133 

(6)  Green  plants. 

1.  Injurious  action  as  indicated  by  retarded  rate 

and  percentage  of  seed  germination  and  by 
retarded  rate  of  plant  growth. 

2.  Beneficial  action  as  shown  by  increased  rate  and 

percentage  of  seed  germination  and  increased 
rate  and  amount  of  plant  growth. 

3.  Modified  in  form,  color,  and  other  "qualitative" 

changes. 

Hypotheses  to  Account  for  Observed  Phenomena.— A  number  of 
hypotheses  have  been  formulated  to  account  for  the  increased  plant 
growth  and  for  the  many  changes  produced  in  soils  by  treatment 
with  heat  and  volatile  antiseptics.  A  number  of  these  theories 
are  considered,  but  it  must  be  borne  in  mind  that  there  is  a  wide 
disagreement  among  workers  as  to  the  real  cause.  No  single 
hypothesis  yet  formulated  can  be  said  to  fully  account  for  all  of  the 
observed  phenomena. 

Koch's  "Direct  Stimulation"  Theory.— The  first  theory  offered  to 
account  for  the  increased  yield  obtained  from  soils  treated  with  an 
antiseptic  was  the  "direct  stimulation"  theory  advanced  by  Koch 
in  1899.  He  considered  carbon  bisulphid  to  have  a  direct  stimulat- 
ing effect  on  the  plants  themselves.  He  later  found  ether  to  have 
a  similar  effect.  In  experiments  dealing  with  the  addition  of  ether 
to  the  soil  Koch  found  that  the  increased  yield  was  pronounced  on 
the  first  crop,  whereas  the  residual  effect  was  slight,  as  with  carbon 
bisulphid  the  beneficial  effect  increases  with  the  amount  of  applica- 
tion. He  further  found  that  soils  sterilized  with  heat  produced 
better  crops  when  treated  with  carbon  bisulphid  than  when  not  so 
treated  and  concludes  that  the  effect  of  the  antiseptic,  therefore, 
cannot  be  due  to  its  effect  on  bacteria. 

The  theory  of  Koch  has  been  supported  by  Fred  who  fertilized 
soil  with  an  abundant  supply  of  sodium  nitrate  and  found  that  in 
every  case  in  which  carbon  bisulphid  was  added  the  growth  and 
yield  of  crop  were  much  superior  to  those  in  the  corresponding  pots 
not  treated  with  that  substance.  He  concludes  that  as  there  was  no 
lack  of  plant-food  and  other  conditions  were  favorable  to  plant 
growth,  the  effect  of  the  antiseptic  must  have  been  directly  upon  the 
plant.  There  is  ample  evidence  to  prove  that  many  of  these  anti- 
septics in  dilute  solutions  stimulate  the  plants  directly,  yet  there 
is  no  evidence  which  will  substantiate  the  claim  that  this  is  the  only 
or  even  the  principal  influence  on  the  plant  and  soil. 

Hiltner  and  Stormer's  "indirect"  theory  of  antiseptic  action  is 
outlined  by  them  as  follows: 

"1.  By  destroying  the  existing  bacterial  equilibrium  in  the  soil, 
the  carbon  bisulphid  opens  the  way  for  an  entirely  new  bacterial 
development.  This  is  achieved  through  the  unequal  retardation 


134  EFFECT  OF  HEAT  ON  SOIL  BACTERIA 

in  the  growth  of  the  different  groups  of  bacteria.  Hence,  certain 
groups  become  disproportionately  prominent,  while  others  are 
almost  entirely  suppressed. 

"2.  The  rapid  increase  in  the  numbers  of  the  bacteria  is  followed 
by  a  more  intense  transformation  of  plant-food  substances.  Decom- 
position and  fixation  processes  result  in  an  accumulation  of  readily 
available  nitrogen  compounds  utilized  by  the  crops.  Hence,  the 
action  of  carbon  bisulphid  is  in  the  nature  of  nitrogen  action. 

"3.  The  initial  suppression  of  the  nitrifying  species  becomes  of 
advantage  in  that  the  nitrogen  compounds,  simplified  by  other 
species,  are  prevented  from  being  rapidly  changed  into  nitrates 
and  being  leached  out  of  the  soil. 

"4.  The  more  or  less  permanent  suppression  of  the  denitrifying 
organisms  must  be  regarded  as  an  additional  factor  favoring  plant 
growth. 

"The  introduction  of  the  poison  into  the  soil  at  first  decimates 
its  bacterial  flora,  but  with  the  disappearance  of  the  injurious 
carbon-bisulphid  vapors  it  also  encourages  a  vigorous  and  long- 
continued  increase  of  the  organisms,  resulting  in  an  increase  of  the 
store  of  more  readily  available  nitrogen.  It  is  still  to  be  deter- 
mined whether  this  increase  is  largely  due  to  the  fixation  of  atmos- 
pheric nitrogen  or  to  the  unlocking  of  the  vast  store  of  combined 
nitrogen  in  the  soil.  It  is  most  probable,  however,  that  even  though 
one  of  these  processes  predominates  the  other  is  surely  more  exten- 
sive than  it  would  be  in  normal  soil.  The  nitrogen  thus  secured  is 
not  at  once  made  accessible  to  the  higher  plants,  but  is  at  first  laid 
fast  in  the  bacterial  bodies.  This  assumption  would  best  explain 
the  fact  that  plants  growing  upon  a  soil  treated  with  carbon  bisulphid 
show  retarded  growth,  even  some  time  after  the  application  of  the 
latter,  and  the  explanation  hitherto  accepted  that  the  injury  results 
from  the  direct  action  of  the  poison  seems  hardly  reasonable  after 
our  discovery  that  the  most  intense  bacterial  activities  are  asserting 
themselves  just  at  that  time.  The  nitrogen  fixed  in  the  bacterial 
bodies  is  gradually  rendered  soluble  by  decomposition  processes, 
and  thereby  made  accessible  to  nitrification  and  the  higher  plants. 
Hence,  when  the  carbon  bisulphid  is  applied  in  the  fall,  there  is 
enough  time  left  until  the  planting  of  the  following  spring  crop  for 
the  mineralization  of  the  bacterial  nitrogen.  It  is  quite  evident,  of 
course,  that  the  nitrogen  combined  in  the  bodies  of  generations  of 
bacteria  is  not  all  made  soluble  within  a  single  year,  but  only  in  the 
course  of  several  growing  seasons,  so  that  we  may  readily  account 
for  the  increased  harvests  secured  for  two  or  more  successive  years 
after  strong  applications  of  carbon  bisulphid,  even  though  the  bac- 
terial transformations  had  by  that  time  declined.  The  exhaustion 
of  the  soil  finally  manifesting  itself  after  a  shorter  or  longer  time 
may  be  explained  by  the  deep-seated  changes  in  the  bacterial  soil 


RUSSELL  AND  HUTCHINSON'S  PROTOZOAN  THEORY     135 

flora,  which  does  not  return  so  easily  to  its  normal  state.  It  is  quite 
possible  that  the  return  to  the  normal  conditions  is  prevented  by 
the  exhaustion  for  years  to  come  of  the  more  available  portions  of  the 
plant  nutrients." 

Evidence  corroborating  this  theory  has  been  brought  forward  by 
Heinze,  Stoklasa,  Lipman,  and  Brown,  whereas  Sirker  furnishes 
evidence  in  the  cultivation  of  the  mulberry  which  opposes  it.  He 
found  that  the  addition  of  carbon  bisulphid  to  a  completely  fertilized 
mulberry  plant  increases  the  vegetation  44  per  cent.,  whereas  a 
heavy  application  of  sodium  nitrate  was  of  slight  value. 

Russell  and  Hutchinson's  Protozoan  Theory.— They  consider  that 
the  microscopic  flora  of  the  ordinary  arable  soil  includes  a  wide 
variety  of  organisms  performing  very  different  functions  which 
may  be  divided  roughly  into  two  classes:  (a)  saprophytes,  tending 
to  increase  the  fertility  of  the  soil,  for  example,  producing  ammonia, 
fixing  nitrogen,  and  similar  changes;  and  (6)  phagocytes  and  large 
organisms  inimical  to  bacteria  which  limit  fertility.  Between  these 
two  classes  of  organisms  there  is  an  equilibrium  under  natural 
conditions,  but  when  partial  sterilization  takes  place  the  phago- 
cytes are  killed  but  the  bacterial  spores  are  not;  and  subsequently 
the  latter  develop  with  great  rapidity,  since  they  are  freed  from  the 
attacks  of  their  enemies,  and  there  is  an  increase  not  only  in 
ammonia  but  likewise  in  crop  production. 

In  support  of  this  theory  they  point  out  the  following:  "In 
untreated  soil  there  is  no  accumulation  of  ammonia,  whereas  the 
'toluene  evaporated'  soil,  as  well  as  the  soil  heated  to  98°  C.,  show 
an  increased  production  of  ammonia.  That  this  is  mainly  the  work 
of  microorganisms  is  proved  by  the  following  considerations:  (a) 
The  curves  belong  to  the  type  associated  with  bacterial,  rather  than 
with  purely  chemical  activities.  (6)  Soil  which  has  been  heated  to 
125°  C.  (at  which  temperature  all  organisms  are  killed)  behaves 
altogether  differently;  after  the  first  production  of  ammonia  due 
to  heating  there  is  no  further  change,  (c)  If  the  toluene  is  left  in 
the  soil  there  is  only  a  slow  production  of  ammonia,  and  never  a 
rapid  rate;  the  curve  is  more  nearly  linear.  The  action  of  micro- 
organisms is  here  excluded,  but  enzymes  may  still  act.  (d)  The 
rapid  period  sets  in  only  when  the  soil  is  sufficiently  moist.  Thus 
the  two  significant  changes  induced  by  partial  sterilization  are, 
(1)  an  increase  in  the  amount  of  ammonia;  and  (2)  cessation  of  the 
nitrifying  process. 

"It  now  becomes  necessary  to  determine  the  part  played  by 
bacteria,  and  why  they  can  increase  so  much  more  rapidly  in  the 
partially  sterilized  soil  (which  accounts  for  the  increased  ammonia 
production)  than  in  the  untreated  soils.  That  the  comparative 
inertness  of  the  bacteria  in  the  untreated  soil  cannot  be  caused  by 
any  bacterial  factor  is  evidenced  by  the  following  considerations: 


136  EFFECT  OF  HEAT  ON  SOIL  BACTERIA 

(a)  If  a  filtered  soil  extract  containing  bacteria  from  an  untreated 
soil  is  added  to  a  toluened  soil,  there  is  an  increase  in  the  rate  of 
ammonia  production,  and  also  in  the  number  of  bacteria.  (6) 
However,  if  untreated  soil  is  added  to  toluened  soil,  there  is  no 
increase,  but  on  the  contrary  a  reduction,  (c)  As  pointed  out  above, 
an  extract  of  the  toluened  soil  is  more  active  than  an  extract  of 
untreated  soil,  (d)  But  when  the  extract  of  toluened  soil  is  added 
to  the  untreated  soil  there  is  no  increase  in  ammonia  production. 

"The  conclusion  drawn  is  that  'the  untreated  soil  contains  a 
factor,  not  bacterial,  limiting  the  development  of  bacteria,  this 
factor  being  put  out  of  action  by  toluening  or  heating.' 

"  Having  determined  the  presence  of  a  limiting  factor  in  untreated 
soils  an  examination  of  its  nature  reveals  that :  (a)  it  is  not  a  toxin, 
for  if  it  were  it  would  be  sure  to  affect  the  nitrifying  bacteria  most; 
(6)  barley  seedlings  grown  in  aqueous  extracts  of  untreated  and 
toluened  soils  showed  no  difference  in  growth  over  a  period  of  four 
weeks;  (c)  the  limiting  factor  is  probably  biological,  for  when 
untreated  soil  is  added  to  toluened  soil  the  reduction  in  the  rate  of 
ammonia  is  not  at  once  operative.  It  is  also  a  large  organism,  since 
it  is  only  in  the  soil  and  not  the  filtered  extract  of  the  untreated  soil 
that  is  effective  in  reducing  the  rate  of  ammonia  production  in 
toluened  soil.  An  examination  of  treated  and  untreated  soil  was 
made,  and  the  latter  revealed  the  presence  of  large  organisms, 
protozoa,  etc.,  which  constitute  the  factor,  or  one  of  the  factors, 
limiting  the  bacterial  activity,  and  therefore  the  fertility  of  untreated 
soil.  Direct  evidence  is  furnished  by  inoculating  toluened  soil  or 
soil  extract  with  cultures  of  large  organisms  and  studying  the 
effect  produced— which  is  a  consequent  depression  in  the  rate  of 
ammonia  formation." 

Although  accepted  by  many  workers,  there  are  many  of  what 
appear  to  be  fatal  objections  that  have  been  brought  against  this 
theory:  (1)  It  has  been  demonstrated  that  the  soil  contains  many 
species  of  fungi  which  are  capable  of  producing  considerable  quan- 
tities of  ammonia  and  these  would  withstand  the  actions  of  the  anti- 
septic or  partial  sterilization  by  heat  and  may  develop  later  and 
produce  large  quantities  of  ammonia.  (2)  There  may  be  a  great 
difference  in  the  physiological  efficiencies  of  the  surviving  ammoni- 
fiers.  (3)  The  work  of  Russell  and  Hutchinson  does  not  consider 
the  probability  of  the  protozoa  being  in  soil  as  cysts.  (4)  The 
direct  laboratory  work  of  Fred  and  Gainey  cannot  be  interpreted  in 
the  light  of  this  theory.  Kopeloff  and  Coleman  analyze  the  work 
of  Fred  in  the  light  of  the  protozoan  theory  as  follows: 

"In  order  to  test  the  validity  of  Russell  and  Hutchinson's  con- 
clusion that  the  absence  of  protozoa  (by  treatment  with  toluene) 
is  responsible  for  increased  production  of  ammonia,  Fred,  using 
ether  instead  of  toluene,  subjected  one  series  of  flasks  containing 


RUSSELL  AND  HUTCHINSON'S  PROTOZOAN  THEORY    137 

compost  soil  to  100°  C.  moist  heat  for  an  hour  and  used  a  similar 
series,  unheated,  as  a  check.  All  the  flasks  received  0.2  per  cent, 
ammonium  sulphate— some  of  the  flasks  received  2  per  cent,  and  5 
per  cent,  ether.  In  order  to  obtain  vigorous  nitrification  170  c.c. 
of  amebae-free  extract  was  inoculated  into  all  the  flasks.  (This 
was  prepared  by  leaching  2  kg.  of  compost  soil  and  4  liters  of  sterile 
water  and  filtering  through  filter  paper;  the  microscopic  examination 
revealed  the  presence  of  no  amebse.) 

"The  analyses  for  nitrate  nitrogen  were  made  at  the  beginning 
of  the  experiments  and  at  the  end  of  100  and  150  days,  respectively; 
the  results  showed  that  heating  the  soil  to  remove  amebse  did  not 
have  a  beneficial  effect  upon  nitrate  formation,  contrary  to  Russell 
and  Hutchinson's  work— although  the  addition  of  a  small  amount 
of  ether  increased  nitrification  in  the  flasks  containing  amebse,  and 
had  the  opposite  effect  in  the  soil  free  from  amebse.  This,  the 
authors  believe,  may  be  accounted  for  by  the  stimulating  effect 
upon  the  nitrifying  bacteria,  since  the  heated  soil  not  treated  with 
ether  showed  no  such  increase. 

"Fred  concludes  (in  addition  to  the  above  mentioned  observa- 
tions) that  ether  and  carbon  bisulphid  cause  an  increased  fixation  of 
nitrogen  in  pure  cultures  of  Azotobacter.  The  development  of  deni- 
trifying organisms  is  hindered  for  only  a  short  time,  because  of 
treatment  with  antiseptics.  Both  Azotobacter  and  denitrifying 
organisms  are  insignificant  in  a  normal  soil.  Nitrification  is  at  first 
inhibited  and  later  accelerated  by  antiseptics,  while  toxins  remain 
unaffected  by  treatment.  '  Carbon  bisulphid  and  ether  cause  an 
increase  in  crop  yield  under  sterile  conditions. 

"He  holds  that  the  increased  growth  of  plants  following  the  use 
of  antiseptics  in  the  soil  depends  essentially  upon  the  stimulation 
to  the  plant  itself,  in  combination  with  a  similar  effect  on  the  lower 
organisms. 

"Fred's  work  is  highly  suggestive,  but  the  determination  of 
nitrogen  produced  is  in  the  form  of  nitrates  alone,  and  no  data  are 
set  forth  concerning  ammonia.  That  this  might  affect  his  results  is 
evident  when  one  takes  into  consideration  the  fact  that  most  investi- 
gators have  proved  that  nitrification  is  depressed  by  antiseptics. 

"  Furthermore,  like  many  other  experimenters  he  does  not  con- 
sider the  possibility  of  protozse  cysts  passing  through  the  filter 
paper  in  the  preparation  of  'amebse-free  extract.'  And  we  have 
found  in  our  experimental  work  that  cysts  do  pass  through  several 
thicknesses  of  high-grade  filter  paper. 

"  In  much  the  same  manner,  Gainey  concludes  that  investigations 
relative  to  the  effect  of  toluol  and  carbon  bisulphid  upon  the  micro- 
flora  and  fauna  of  the  soil,  that:  (a)  small  quantities  of  carbon 
bisulphid,  toluol,  and  chloroform,  such  as  have  been  used  practically 
and  experimentally,  when  applied  to  soils  studies,  exert  a  stimulative 


138  EFFECT  OF  HEAT  ON  SOIL  BACTERIA 

rather  than  a  diminishing  effect  upon  the  total  number  of  bacteria 
present;  (6)  an  application  of  such  quantities  of  toluol  and  carbon 
bisulphid  does  not  have  an  appreciable  effect  upon  the  number  of 
types  of  protozoa  present;  (c)  a  very  marked  increase  in  yield  may 
be  noted  following  such  an  application  when  no  evident  change 
occurs  in  the  total  number  of  bacteria  present." 

Greig-Smith's  Bacteriotoxin  Theory.— A  widely  different  theory 
from  any  so  far  considered  is  that  advanced  by  Greig-Smith.  He 
considers  that  when  disinfectants  are  added  to  the  soil  their  action 
is  two-fold:  They  kill  the  less  resistant  bacteria  and  dissolve  from 
the  surface  of  the  soil  particles  a  waxy  covering  to  which  he  has  given 
the  name  "agricere."  The  surviving  bacteria,  which  he  assumes 
are  the  beneficial  ones,  are  then  able  to  function  much  more  rapidly 
on  account  of  the  exposure  of  the  food  due  to  the  removal  of  the 
"agricere." 

Moreover,  he  considers  that  there  is  a  toxin  contained  in  the  soil 
which  is  soluble  in  dilute  saline,  partially  destroyed  by  heating  to 
94°  C.,  and  rapidly  decayed  in  aqueous  solution;  boiling  water 
converts  it  into  a  nutrient,  or  by  destroying  the  toxin  enables  the 
nutrients  dissolved  in  the  saline  to  act.  Thus,  heating  the  soil 
destroys  the  bacteria  toxin,  which  accounts  for  enhanced  fertility. 
Bottomley  and  others  also  claim  to  have  found  soluble  bacterio- 
toxins  in  soils.  Russell  and  Hutchinson,  on  the  other  hand,  obtained 
wholly  negative  results,  and  conclude  that  soluble  bacteriotoxins 
are  not  normal  constituents  of  soils,  but  must  represent  unusual 
conditions  wherever  they  occur.  Not  only  could  no  experimental 
evidence  of  the  existence  of  bacteriotoxins  be  obtained,  but  Russell 
and  Thaysen  showed  that  the  assumption  of  toxins  leads  to  difficul- 
ties. It  is  necessary  to  suppose  that  heating  fresh  soil  for  fifteen 
minutes  is  sufficient  to  produce  toxins  but  not  to  destroy  them, 
whereas  heating  for  sixty  minutes  both  produces  and  destroys  them, 
and  in  the  case  of  air-dried  soils  fifteen  minutes'  heating  causes  their 
decomposition. 

REFERENCES. 

Vorhees,  E.  B.  and  Lipman,  J.  G.:  A  Review  of  Investigations  in  Soils  Bacteri- 
ology (U.  S.  D.  A.  Off.  Exp.  Sta.  Bui.  194). 

Lohnis:     Handbuch  der  Landwirtschaftlichen  Bakteriologie. 

Kopeloff,  Nicholas  and  Coleman,  D.  A.:  A  Review  of  Investigations  in  Soil  Pro- 
tozoa and  Soil  Sterilization,  Soil  Science,  1917,  iii,  197-269. 

Johnson,  James:  The  Influence  of  Heated  Soils  on  Seed  Germination  and  Plant 
Growth,  Soil  Science,  1919,  vii,  1-103. 


I 


CHAPTER  XIV. 

THE  INFLUENCE  OF  SALTS  ON  THE  BACTERIAL 
ACTIVITIES  OF  THE  SOIL. 

SALTS  that  occur  naturally  in  soils  and  those  applied  to  them  in 
various  operations  influence  the  number,  species,  and  activity  of 
the  soil  microflora.  These  factors  are  in  turn  reflected  by  yields 
obtained.  Some  substances  applied  to  a  soil  serve  as  food  for  the 
growing  plant;  others  increase  plant  growth  but  not  through  the 
direct  furnishing  of  food.  This  latter  effect  may  be  due  to  a  change 
brought  about  by  the  salt  on  the  physical,  chemical,  or  bacterial 
properties  of  the  soil.  The  substance  may  alter  the  physical  proper- 
ties of  the  soil  to  such  an  extent  that  the  bacterial  flora  is  modified ; 
this  in  turn  may  increase  or  decrease  the  crop  produced  upon  the 
soil.  Other  substances  may  react  chemically  with  constituents 
within  the  soil  and  in  so  doing  liberate  substances  which  can  be 
directly  utilized  by  the  growing  plant.  Again,  they  may^directly 
modify  the  microflora  and  microfauna  of  the  soil  both  as  to  numbers 
and  physiological  efficiency.  In  some  cases  all  three  changes  may 
be  wrought  by  the  same  salt.  The  question,  therefore,  arises  as  to 
what  effect  this  or  that  fertilizer  or  soil  amendment  is  going  to  have 
upon  the  bacterial  activity  of  the  soil.  Furthermore,  there  are 
millions  of  acres  of  land  in  arid  America  which  contain  varying 
amounts  of  soluble  salts.  Some  of  these  soils  contain  such  large 
quantities  of  these  so-called  "alkalies"  that  no  vegetation  is  found 
upon  them.  Other  soils  contain  only  a  medium  amount  of  soluble 
salts  and  the  vegetation  is  composed  chiefly  of  alkali-resisting  plants. 
Still  other  soils  contain  much  smaller  quantities  of  soluble  salts  and 
they  become  injurious  only  when  the  soil  is  improperly  handled. 
The  reclaiming  of  the  heavily  charged  soils  and  the  maintaining  of 
the  others  in  a  productive  condition  can  be  carried  on  successfully 
only  when  we  understand  the  influence  of  salts  upon  the  growing 
plants  and  their  action  upon  the  biological,  chemical,  and  physical 
properties  of  the  soil. 

Calcium  Carbonate.— Much  work  has  been  done  to  determine  the 
influence  of  calcium  carbonate,  especially  when  applied  to  acid  soils, 
on  the  bacterial  content  and  activity  of  the  soil,  but  the  conclusions 
reached  have  not  always  been  concordant.  Withers  and  Fraps 
found  that  calcium  carbonate  added  to  a  soil  greatly  accelerated 
nitrification  and  that  it  is  especially  desirable  that  it  should  be 


140  INFLUENCE  OF  SALTS  ON  THE  SOIL 

added  where  ammonium  sulphate  is  being  used  as  a  fertilizer.  Lip- 
man's  work  showed  that  calcium  carbonate  stimulated  nitrification 
more  than  did  gypsum,  that  sodium  chlorid  was  injurious  to  nitrify- 
ing organisms,  and  that  ferrous  sulphate  in  amounts  from  10  to  100 
mg.  per  100  gm.  of  soil  was  without  effect.  Later,  he  and  Brown 
decided  that  both  ammonification  and  nitrification  were  promoted 
by  magnesia  lime  to  a  more  marked  extent  than  they  were  by  non- 
magnesia  lime.  This,  however,  was  to  a  certain  extent  dependent 
upon  the  treatment  and  crop  growing  on  the  soil.  Both  ammonifi- 
cation and  nitrification  were  accelerated  by  sodium  nitrate.  In  a 
more  recent  work  Lipman,  Brown,  and  Owen  found  that  small 
applications  of  calcium  carbonate  stimulated  bacterial  activity, 
whereas  large  applications  had  a  detrimental  effect  upon  ammonifi- 
cation. 

In  Owen's  experiments,  magnesium  carbonate  was  more  efficient 
in  promoting  ammonification  and  nitrification  than  was  either 
calcium  or  potassium  carbonate.  According  to  Engberding  ammo- 
nium sulphate,  sodium  nitrate,  potassium  nitrate,  and  caustic  lime 
all  increase  the  bacterial  content  of  the  soil,  but  decrease  its  nitrogen- 
fixing  powers.  Kruger's  work  indicated  that  calcium  carbonate 
was  more  effective  in  promoting  nitrification  than  was  lime,  the 
reverse  being  true  with  regard  to  the  putrefactive  bacteria.  The 
formation  of  ammonia  from  peptone  was  especially  favored  by 
calcium  carbonate.  Lyon  and  Bizzell  found  that  lime  favored 
nitrification,  as  did  also  certain  nodule-bearing  legumes.  Fischer 
concluded  that  the  presence  of  calcium  carbonate  in  a  nutritive 
solution  favored  the  formation  of  protein  nitrogen,  but  magnesium 
carbonate  lessened  the  transformation  of  ammonia  into  protein 
nitrogen.  Calcium  oxid,  however,  exerted  a  much  greater  influence 
upon,  soil  bacteria  than  did  calcium  carbonate. 

Kellermann  and  Robinson's  results  are  of  especial  interest  as 
they  indicate  that  magnesium  carbonate,  applied  in  amounts 
exceeding  0.25  per  cent,  to  a  soil  fairly  high  in  magnesium  carbonate, 
positively  inhibited  nitrification,  whereas  calcium  carbonate  up  to 
2  per  cent,  favored  it,  thus  indicating  that  the  lime-magnesia  ratio 
is  of  great  importance  with  regard  to  bacteria  as  well  as  the  higher 
plants.  These  results  have  been  confirmed  by  C.  B.  Lipman  and 
Burgess  in  whose  experiments  magnesium  carbonate  was  highly 
toxic  both  in  soil  and  in  solution  to  Azotobacter  chroococcum,  while 
calcium  carbonate  was  never  toxic  even  in  quantities  up  to  2  per 
cent.  Furthermore,  calcium  carbonate  exerted  a  protective  influ- 
ence against  the  toxic  properties  of  magnesium  carbonate.  The 
optimum  ratio  varied,  depending  upon  the  medium. 

Peck  studied  the  influence  of  a  number  of  salts  upon  bacterial 
activity  when  applied  to  the  soil,  with  the  result  that  the  carbonate, 
sulphate,  and  phosphate  of  calcium  were  found  to  stimulate  ammoni- 


LIME  141 

fication,  while  sodium  nitrate  depressed  it;  both  potassium  sulphate 
and  calcium  carbonate  accelerated  nitrification  in  soil.  Brown, 
working  with  a  typical  Wisconsin  drift  soil,  found  that  the  applica- 
tion of  ground  lime  up  to  3  tons  an  acre  increased  the  number  of 
bacteria  in  the  soil,  and  also  the  ammonifying,  nitrifying,  and  nitro- 
gen-fixing powers  of  the  soil.  The  increase  was  in  every  case  nearly 
proportional  to  the  limestone  applied. 

At  times  the  increase  noted  in  ammonification  is  due  to  the 
retention  of  the  volatile  ammonia  by  the  carbonate,  as  is  shown  by 
Lemmermann's  results  where  the  addition  of  calcium  carbonate  to  a 
soil  up  to  1  per  cent,  reduced  the  volatilization  of  ammonia,  but 
calcium  oxid  had  the  opposite  effect.  Both  calcium  chlorid  and 
calcium  sulfate  reduced  the  loss  of  ammonia,  but  the  chlorid  was 
the  only  salt  of  magnesium  tested  which  had  this  effect.  Potassium 
and  sodium  chlorids,  sulphates,  and  carbonates  all  reduced  the 
absorptive  powers  of  the  soil.  Paterson  studied  the  influence  of  a 
number  of  substances  upon  nitrification  with  the  result  that  caustic 
lime  was  found  practically  to  stop  all  nitrification.  Calcium  car- 
bonate promoted  it,  as  did  also  magnesium  carbonate;  gypsum  was 
lesfe  effective,  while  ferric  hydrate  had  a  decidedly  favorable  effect. 
Sodium  chlorid,  on  the  other  hand,  had  a  distinctly  injurious  effect. 

Kelley  studied  the  effect  of  calcium  and  magnesium  carbonate 
alone  and  in  combination  upon  ammonification  and  nitrification. 
In  his  work  calcium  carbonate  only  slightly  stimulated  ammonifi- 
cation of  dried  blood,  but  it  had  a  marked  stimulating  effect  upon 
nitrification.  The  magnesium  carbonate  was  found  to  be  toxic 
to  both  groups  of  organisms.  No  antagonism  was  found  to  exist 
between  calcium  and  magnesium.  Later,  when  working  with 
Hawaiian  soils,  he  reports  a  stimulation  for  both.  The  results, 
however,  varied  with  different  soils,  and  he  considers  the  lime- 
magnesia  ratio  of  little  importance  as  regards  the  ammonifying 
and  nitrifying  organisms.  Allen's  conclusion  is  that  large  quantities 
of  limestone  must  be  applied  to  a  non-calcareous  soil  in  order  to 
bring  its  nitrifying  powers  up  to  those  of  natural  calcareous  soils. 

Lime.— Peterson  and  Wollny  found  that  lime  increased  the  carbon 
dioxid  given  off  by  soils,  and  Ebermayer,  Hilgard,  and  Hart  well 
and  Kellogg  proved  conclusively  that  lime  increases  the  decay  taking 
place  in  a  soil. 

Chester  showed  that  lime  increased  the  number  of  bacteria  in 
soil,  the  increase  being  proportional  to  the  lime  applied  up  to  4000 
pounds  an.  acre.  He  considered  the  effect  as  being  due  to  the  lime 
giving  to  the  soil  a  more  favorable  reaction  for  the  growth  of  bac- 
teria and  not  to  its  direct  action  upon  the  organisms  themselves. 

Lime  not  only  increases  the  number  of  organisms  in  a  soil,  but 
it  increases  the  ammonifying  powers  of  the  soil,  as  is  seen  from  the 
work  of  Remy,  Ehrenberg,  Vorhees,  and  Lipman, 


142  INFLUENCE  OF  SALTS  ON  THE  SOIL 

The  literature  dealing  with  the  influence  of  lime  upon  the  nitrify- 
ing organisms  is  so  voluminous  that  no  attempt  is  made  here  to 
refer  to  all  of  it.  In  most  cases  the  experiments  were  conducted  on 
soils  which  were  acid  and  the  lime  supplied  neutralized  the  acidity 
of  the  soil,  thus  giving  the  necessary  neutral  medium  for  the  action 
of  the  nitrifying  organisms.  Such  results  give  little  if  any  idea  of 
the  direct  stimulating  or  toxic  influence  of  calcium,  Furthermore, 
the  work  has  recently  been  summarized  by  Brown  who  concludes 
that  the  application  of  lime  increased  nitrate  production  from 
ammonium  sulphate  and  dried  blood,  the  gain  being  almost  propor- 
tional to  the  quantity  of  lime  applied.  This,  in  turn,  was  found  to 
bear  a  close  relationship  to  the  number  of  organisms  developing  on 
synthetic  agar. 

Gypsum.— Gypsum  is  a  strong  soil  stimulant  and  in  most  cases  it 
greatly  increases  the  crop  yield.  The  beneficial  effect  may  be  due 
to  its  liberating  potassium  or  supplying  sulphur  for  the  direct  nutri- 
tion of  the  plant.  At  other  times  it  may  react  with  the  ammonia 
formed  by  the  ammonifying  organisms,  the  ammonium  sulphates 
formed  being  readily  nitrified.  There  is  also  the  possibility  that 
calcium  sulphate  acts  as  a  direct  stimulant  to  the  microorganisms  of 
the  soil.  The  literature  is  meager  and  inconclusive  on  this  phase 
of  the  subject. 

Severin  concludes  from  his  work  that  gypsum  not  only  prevents 
the  loss  of  ammonia  from  manure,  but  it  increases  the  speed  of 
decomposition  from  10  to  20  per  cent.,  while  Paterson  states  that 
gypsum  slightly  increases  nitrification  in  soil,  as  determined  by 
laboratory  experiments.  Prior  to  this  Pichard  had  shown  that  the 
sulphates  of  calcium,  potassium,  and  sodium  promote  nitrification. 

Opposite  results  are  reported  by  Dezani  who  found  that  gypsum 
in  amounts  varying  from  0.5  to  2  per  cent,  had  no  appreciable 
effect  on  nitrification.  The  results  obtained  by  Lipman  and  others 
varied  and  were  inconclusive. 

Calcium  Chlorid.  —According  to  Lipman  calcium  chlorid  in  solutions 
accelerated  the  action  of  ammonifiers.  It  is  interesting  to  note  that 
in  a  later  work  he  failed  to  find  antagonism  between  either  calcium 
and  magnesium  or  calcium  and  sodium.  The  chlorid s  of  calcium, 
magnesium,  potassium,  and  sodium  were  found  to  be  toxic  in  the 
order  named.  Marked  antagonism  exists  between  calcium  and 
potassium  magnesium  and  sodium,  and  potassium  and  sodium. 
Sea  water  was  found  to  be  a  physiologically  balanced  solution  for 
Bacillus  subtilis. 

Iron  Sulphate. — Many  writers  have  made  great  claims  for  iron 
sulphate  as  a  fertilizer.  A  goodly  number  of  these  have  been  made 
by  individuals  who  wished  to  profit  by  its  sale,  but  even  when  these 
cases  are  ignored  there  are  still  many  cases  in  which  it  has  produced 
good  results.  The  composition  of  the  crop  usually  indicates Vthat 


MANGANESE  143 

iron  sulphate  influences  the  phosphorus  metabolism  of  the  plant. 
It  is  hard  to  see  how  this  is  possible  unless  it  be  that  the  iron  stimu- 
lates the  bacterial  activity,  which  in  turn  liberates  phosphorus 
from  its  insoluble  forms  within  the  soil.  It  has  already  been  noted 
that  Lipman  found  ferrous  sulphate  to  have  but  little  effect  upon 
nitrification,  but  his  results  were  not  conclusive.  Guffroy  found 
sulphate  of  iron  decidedly  beneficial  to  oats,  less  beneficial  to  rye, 
without  action  on  rye-grass,  and  harmful  to  wheat.  He  concluded 
that  its  action  must  be  due  to  its  influence  on  the  biological  processes 
of  the  soil.  According  to  Paterson  and  Scott,  ferric  hydrate  has  a 
distinctly  beneficial  effect  upon  nitrification.  In  this  latter  case 
its  action  could  be  due  to  its  serving  as  a  base.  According  to 
Lipman  and  Burgess  the  ammonifiers  are  more  sensitive  to  iron 
sulphate  than  are  the  nitrifiers,  for  though  small  amounts  of  iron 
sulphate  stimulated  the  latter,  it  was  toxic  to  the  former  in  all  con- 
centrations tested. 

Magnesium  Salts.— Magnesium  compounds  usually  stimulate 
bacterial  activities  to  a  greater  extent  than  do  calcium  compounds, 
as  has  been  noted  in  some  of  the  literature  already  cited,  Eng- 
berding's  results,  however,  showed  that,  while  magnesium  sulphate 
stimulated  bacterial  activities,  it  was  not  as  effective  in  this  regard 
as  was  ammonium  sulphate,  sodium  nitrate,  or  potassium  sulphate. 
The  work  of  Makrinov  is  of  interest  since  he  found  pure  magnesium 
carbonate  a  very  suitable  substance  on  which  to  grow  the  nitrous 
organism.  Furthermore,  magnesium  carbonate  had  a  strongly 
beneficial  effect  on  the  physiological  action  of  the  organism.  Keller- 
mann  and  Robinson,  on  the  other  hand,  found  that  magnesium  car- 
bonate when  applied  to  a  soil  already  rich  in  magnesium  carbonate 
positively  inhibited  nitrification  if  the  quantity  added  exceeded 
0.25  per  cent.  This  is  an  apparent  contradiction,  but  it  may  be 
due  to  the  different  conditions  of  the  experiments,  since  one  investi- 
gator was  working  with  culture  of  the  organisms  whereas  the  other 
was  using  the  soil  with  its  complex  flora.  Furthermore,  it  is  quite 
possible  that  magnesium  carbonate  may  be  without  effect  upon  or 
even  accelerate  the  growth  and  activity  of  the  Nitrosomonas  and 
yet  inhibit  the  Nitromonas. 

Manganese.— Some  experiments  by  Skinner  and  Sullivan  demon- 
strate that  manganese  acts  in  various  ways  as  a  fertilizer.  It  is 
often  without  influence,  occasionally  injurious,  but  usually  bene- 
ficial, its  effect  depending  apparently  upon  the  composition  and 
character  of  the  soil.  The  oxidation  in  soils  under  treatment  with 
manganese  salts  was  also  studied  and  it  was  found  that  an  increase 
in  oxidation  and  growth  frequently  occurred  in  aqueous  extracts  of 
poor,  unproductive  soils.  Although  oxidation  was  increased  in 
fertile  soils,  growth  was  decreased,  the  plants  showing  indications 
of  excessive  oxidation.  Field  experiments  showed  practically  no 


144  INFLUENCE  OF  SALTS  ON  THE  SOIL 

effect  due  to  the  manganese  salts,  but  the  soil  was  acid,  a  condition 
which  may  have  accounted  to  a  considerable  degree  for  the  nature 
of  the  results. 

It  is  suggested  that  when  the  action  of  manganese  is  beneficial, 
"it  is  probably  due  (1)  to  the  increased  oxidation  produced  in  the 
plant  roots  whereby  the  plant  is  stimulated  to  greater  activity  and 
to  increased  absorption  of  the  material  useful  for  its  growth  and 
general  metabolism;  (2)  to  the  stimulation  of  the  activity  of  micro- 
organisms in  the  soil;  (3)  to  an  increased  oxidation  within  the  soil." 

The  same  authors  also  suggest  that  when  large  applications  of 
manganese  have  been  found  to  be  injurious,  the  injury  is 
undoubtedly  due  to  the  "excessive  stimulation  and  excessive  oxida- 
tion in  microorganisms  and  in  the  plant,  with  a  resulting  change  in 
the  biochemical  activities  of  plant  and  microorganisms  and  in  the 
conditions  of  inorganic  and  organic  soil  constituents,  the  ultimate 
result  of  which  change  is  injurious  to  the  growing  crop." 

Montanan  found  that  manganese  carbonate,  sulphate,  and  dioxid 
greatly  stimulated  nitrification,  but  attributed  it  to  either  the  direct 
or  the  indirect  furnishing  of  oxygen  to  the  nitrifying  organisms, 
and  not  to  any  catalytic  effect  of  the  manganese  itself.  Beijerinck 
observes  that  some  soil  organisms  have  the  power  of  oxidizing 
manganous  carbonate.  Olaru  found  that  manganese  in  the  propor- 
tion of  one  part  to  200,000  parts  of  nutritive  media  greatly  increases 
nitrogen  fixation.  He  also  considers  it  rather  likely  that  the 
increased  yield  obtained  after  the  application  of  manganese  com- 
pounds to  a  soil  is  due  to  its  accelerating  the  action  of  the  nitrogen- 
fixing  organisms  of  the  soil. 

Leoncini  found  that  the  application  of  manganese  oxid  to  the 
soil  at  the  rate  of  0.035  to  2.2  per  cent,  favored  nitrification,  whereas 
larger  amounts  had  no  effect. 

Brown's  work,  which  is  probably  the  most  extensive  study  of  the 
influence  of  manganese  compounds  on  bacterial  activities  so  far 
reported,  shows  that  the  influence  of  the  manganese  salt  upon  bac- 
terial activities  varies  with  (1)  the  kind  of  salt  added— manganese 
chlorid  and  sulphate  acting  in  low  concentrations  as  stimulants  to 
both  ammonifying  and  nitrifying  bacteria,  but  in  greater  concentra- 
tion being  highly  toxic;  (2)  its  action  varies  with  the  specific  class 
of  organisms  which  were  being  studied,  the  ammonifiers  being  more 
resistant  than  are  the  nitrifiers. 

Potassium  Salts.— Because  potassium  is  essential  for  the  nutrition 
of  both  the  higher  and  lower  forms  of  plant  life,  it  is  to  be  expected 
that  when  added  to  a  soil  medium  poor  in  potassium  it  will  increase 
bacterial  growth,  but,  like  many  other  true  nutrients,  may  become 
toxic  if  present  in  too  high  concentrations.  As  already  noted, 
Engberding  states  that  potassium  sulphate  caused  a  slight  increase 
in  the  bacterial  content  of  a  soil.  While  Peck  found  it  to  increase 


SODIUM  SALTS  .  145 

nitrification  in  soils,  Renault  claims  that  slow  ammonification  and 
subsequent  nitrification  is  always  accompanied  by  a  low  percentage 
of  potash.  Dumont's  experiments  showed  that  potassium  car- 
bonate, added  to  a  soil  at  the  rate  of  from  1  to  2.5  gm.  per  1000  gm. 
of  soil,  markedly  increased  nitrification,  but  that  larger  applications 
of  the  salt  progressively  diminished  the  rate  of  nitrification,  while 
the  addition  of  8  gm.  to  1000  gm.  of  soil  completely  checked  it. 
Lumia  concluded  that  potassium  chlorid  and  sulphate  were  nearly 
as  effective  in  promoting  the  activity  of  alcoholic  ferments  as  were 
phosphates. 

Fred  and  Hart  found  that  both  calcium  and  potassium  sulphates 
increased  ammonification  in  solution  and  that  the  sulphates  of  potas- 
sium, calcium,  and  magnesium  each  increased  the  volume  of  carbon 
dioxid  from  soil.  From  the  results  obtained  with  different  salts, 
they  conclude  that  the  addition  of  the  potassium  ion  did  not  mate- 
rially increase  ammonification  in  the  soil  examined. 

Sodium  Salts.— Sodium  salts  are  often  used  as  fertilizers  and  with 
good  results.  Furthermore,  many  alkali  soils  contain  sodium  salts 
in  quantities  sufficient  to  be  toxic  to  both  the  higher  and  lower 
plants.  For  these  reasons  many  investigations  have  been  conducted 
to  determine  the  influence  of  sodium  compounds  upon  higher 
plants,  and  many  have  had  as  their  object  the  determination  of  their 
influence  upon  soil  bacteria. 

As  early  as  1884  Warington  showed  that  the  presence  of  0.032 
per  cent,  of  sodium  bicarbonate  distinctly  retarded  nitrification, 
and  that  in  the  presence  of  0.096  per  cent,  nitrification  was  very 
slight.  Schlosing  had  added  various  salts  to  the  soil  in  quantities 
not  exceeding  485  parts  per  million  with  no  apparent  effect  upon 
nitrification.  However,  Deherain  found  that  common  salt  com- 
menced to  be  harmful  when  it  exceeded  one-thousandth  of  the  weight 
of  soil,  and  when  larger  quantities -are  applied  nitrification  almost 
ceased.  According  to  the  same  observer  sodium  nitrate  may  stop 
nitrification  for  a  time,  but  later  it  recommences.  Lipman  and 
others  found  that  sodium  nitrate  increased  the  accumulation  of 
nitrates  in  a  soil.  They  found,  however,  a  certain  periodicity  in 
the  accumulation  of  nitrates  which  would  account  for  the  different 
results  reported  by  various  investigators.  In  later  investigators  they 
concluded  that  at  times  sodium  nitrate  stimulates  ammonification. 
McBeth  and  Wright  found  that  carbonates,  chlorids,  and  sulphates 
inhibited  nitrification  and  that  the  former  were  more  injurious  than 
the  latter. 

The  most  far-reaching  and  systematic  work  which  has  been 
reported  on  the  influence  of  salts  upon  bacterial  activity  is  the 
excellent  work  by  C.  B.  Lipman  who  demonstrated  that  ammonifi- 
cation is  inhibited  by  sodium  chlorid,  sodium  sulphate,  and  sodium 
carbonate.  The  points  at  which  the  salts  became  toxic  are:  for 
10 


146  INFLUENCE  OF  SALTS  ON  THE  SOIL 

sodium  chlorid,  between  0.1  per  cent,  and  0.2  per  cent.;  for  sodium 
sulphate,  0.4  per  cent.;  and  for  sodium  carbonate,  2.0  per  cent.  A 
stimulating  influence  was  noted  in  the  case  of  sodium  carbonate, 
but  not  in  the  case  of  the  sulphate  or  the  chlorid.  The  points  at 
which  they  became  toxic  to  nitrifiers  were  found  to  be:  for  sodium 
carbonate,  0.025  per  cent.;  for  sodium  sulphate,  0.35  per  cent.; 
and  for  sodium  chlorid,  less  than  0.1  per  cent. 

All  except  sodium  chlorid  acted  as  a  stimulant  in  lower  concen- 
trations. Later  Lipman  and  Sharp  found  the  point  at  which  sodium 
chlorid  became  toxic  to  nitrogen-fixing  organisms  in  soil  to  be  from 
0.5  to  0.6  per  cent.;  sodium  sulphate,  at  1.25  per  cent.;  and  sodium 
carbonate,  at  0.4  to  0.5  per  cent.  Sodium  chlorid  was  the  only  one 
which  acted  as  a  stimulant.  Recently  Lipman  has  demonstrated 
that  there  exists,  as  measured  by  ammonification,  a  true  antagonism 
between  sodium  chlorid  and  sodium  sulphate;  between  sodium 
chlorid  and  sodium  carbonate;  and  between  sodium  sulphate  and 
sodium  carbonate. 

Brown  and  Hitchcock  found  nitrification  to  be  stimulated  by 
small  amounts  of  sodium  chlorid,  sodium  sulphate,  and  magnesium 
carbonate,  and  large  amounts  of  calcium  carbonate.  The  large 
quantities,  however,  became  toxic,  the  point  at  which  toxicity  and 
probably  stimulation  occurs  varying  with  the  different  soils. 

Variation  in  Effect  Produced.— It  is  quite  evident  from  the  liter^a- 
ture  reviewed  that  the  addition  of  a  salt  to  a  soil  may  produce  a 
number  of  different  effects,  depending  upon  the  nature  and  quantity 
of  the  salt  added:  (1)  The  salt  may  stimulate  some  or  all  of  the 
bacterial  activities  of  the  soil;  (2)  it  may  be  without  effect;  (3)  it 
may  be  toxic  to  some  and  without  effect  or  stimulating  to  others; 
(4)  it  may  be  toxic  to  all  of  the  bacteria  of  the  soil  and  hence  either 
kill  the  organisms  or,  what  is  more  often  the  case,  materially 
decrease  their  metabolic  activity. 

These  factors  are  well  illustrated  in  an  extensive  study  carried 
on  by  the  author  on  the  chlorids,  nitrates,  sulphates,  and  carbonates 
of  sodium,  potassium,  calcium,  magnesium,  manganese,  and  iron. 
They  are  especially  interesting  in  that  it  indicates  the  influence  of 
these  twenty-four  salts  on  the  ammonifying  and  nitrifying  powers  of 
a  soil  all  tested  under  similar  conditions.  Though  the  results  are 
not  absolute,  they  do  represent  rather  nearly  the  relative  action  of 
the  various  salts,  as  may  be  seen  by  comparing  them  with  the 
results  reported  by  others.  One  fact,  however,  which  the  student 
must  bear  in  mind  with  these,  as  with  all  other  results,  is  that  an 
accumulation  of  a  specific  substance  within  the  soil  may  represent 
either  an  acceleration  of  the  activity  of  the  organisms  which  form 
that  compound  or  a  decrease  of  the  efficiency  of  the  organism  which 
Destroys  it, 


STIMULATING  ACTION 


147 


Stimulating  Action.— Only  six  of  the  twenty-four  compounds 
tested  failed  to  stimulate  the  ammonifying  organisms  at  some  con- 
centration. Those  which  did  not  stimulate  were  calcium  chlorid, 


§*T  H-        M  [O       CO  CO       *• 

^>  w      t>  ui     O  m     O 

X  *  *  1  *  *  *'- 

o  S,  S,  B,  °,  °.  °,  °.  °- 


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Cn 

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Na2CO3 
CaCO3 

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K2SO4 

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MnCI2 

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FIG.  20, — Graphs  showing  molecular  concentrations  at  which  the  various  salts  exert 
greatest  stimulation  on  bacteria  in  soil. 


148 


INFLUENCE  OF  SALTS  ON   THE  SOIL 


calcium  nitrate,  potassium  chlorid,  potassium  sulphate,  magnesium 
nitrate,  and  sodium  sulphate. 


FIG.  21. — Graphs  showing  the  percentage  of  stimulation  at  the  above  noted  molec- 
ular concentrations  (See  Fig.  20),  the  untreated  soil  being  counted  as  producing 
100  per  cent,  of  nitric  nitrogen. 


1  x  10' 

rl    7  x  10  *t 

1                                                       6xlO"3, 

6x10^ 

J(                                          4  X  10" 

65  x  10  -,u  ^ 
60  x  10"    [-" 
55  x  10  •    U; 

EXPLANATION 

NITRIFICATION  •••• 

AMMOIFITATinN  1              ] 

50  x  10" 
45x10* 

43x10' 

- 

35  x  10- 

3  J  x  10  ' 

- 

25  x  10  " 
20  x  10  '     ' 

1- 

150  x  10 

n 

- 

100  x  10 
500  x  10'7  " 

3 

I 

r 

• 

1 

0     j 

_r-,          1 

^n  -.     -    _ 

•r-,  —  -,  —  ,  •-!-• 

• 

«-i  mf-i  m 

8     1 

1 

FIG.  22. — Graphs  showing  the  molecular  concentrations  at  which  the  various  salts 
are  toxic  to  ammonifying  and  nitrifying  organisms  in  the  soil. 


There  were  also  six  which  failed  to  stimulate  the  nitrifiers,  but 
it  is  quite  evident  from  the  results  given  in  Fig.  20  that  in  the 
majority  of  cases  these  are  different  from  the  ones  which  failed  to 
stimulate  the  ammonifiers.  This  is  remarkable  when  we  remember 


TOXICITY  OF  VARIOUS  SALTS  149 

that  the  speed  of  the  nitrification  process  is  controlled  and  dependent 
upon  the  speed  of  ammonification.  The  results  clearly  indicate  that 
there  are  other  side  reactions  taking  place  which  are  influenced  by 
the  salts  but  which  are  not  measured  by  the  ordinary  bacterio- 
logical method. 

It  is  evident,  however,  from  the  results  reported  in  Fig.  21  that 
those  compounds  which  are  most  active  as  stimulants  to  the  higher 
plants  are  also  most  active  in  stimulating  bacteria.  It  is  likely  that 
the  effect  upon  the, plant  is  due  in  a  large  measure  to  the  action  of 
the  compound  upon  the  bacteria,  which  in  turn  render  available 
more  plant-food. 

Toxicity  of  Various  Salts.— There  is  an  extremely  wide  variation  in 
the  concentration  at  which  various  salts  become  toxic  to  soil  bac- 
teria (Fig.  22).  Some  must  occur  in  soils  in  large  quantities  before 
becoming  toxic,  whereas  others  are  toxic  when  present  in  only  minute 
quantities.  The  toxicity  of  the  salts  to  ammonifying  organisms  are 
controlled  largely  by  the  electronegative  ion,  but  this  is  not  as 
pronounced  in  the  case  of  the  nitrifiers.  The  latter  class  of  organisms 
is,  however,  more  sensitive  to  salts  than  are  the  ammonifiers.  The 
ammonifiers  represent  more  nearly  the  higher  plant. 

It  is  apparent  from  these  results  that  the  increased  osmotic 
pressure  exerted  by  a  salt  within  the  soil  plays  a  part  in  retarding 
the  bacterial  activity  of  such  a  soil,  but  it  is  not  the  only  factor. 
The  main  factor  is  probable  a  physiological  one,  due  to  the  action 
of  the  substance  upon  the  living  protoplasm  of  the  cell  changing 
its  chemical  and  physical  properties  in  such  a  way  that  it  cannot 
function  normally. 

REFERENCE. 

Greaves,  J.  E.:  "The  Influence  of  Salts  on  the  Bacterial  Activity  of  the  Soil" 
(Soil  Science,  1916,  ii,  443-480). 


CHAPTER  XV. 

INFLUENCE  OF  MANURE  ON  THE  BACTERIAL 
ACTIVITIES  OF  THE  SOIL. 

THE  application  of  barnyard  manure  to  a  soil  brings  about  a 
far-reaching  change  within  the  soil.  It  has  been  found  that,  on  the 
average,  one  ton  of  barnyard  manure  contains  10  or  12  pounds  each 
of  nitrogen  and  potassium  and  2  or  3  pounds  of  phosphorus.  It  also 
carries  other  substances  of  less  importance  which  may  be  directly 
utilized  by  the  growing  plant  or  which  may  react  with  substances 
within  the  soil,  changing  their  solubility.  This  direct  and  indirect 
nutritive  value  of  a  manure  is  not  its  only  function,  for  it  greatly 
changes  the  physical  structure  of  the  soil.  It  improves  the  tilth  of 
a  clay  soil  by  increasing  the  granulation  within  it,  while  in  a  sandy 
soil  it  tends  to  bind  the  particles  together,  making  it  Jess  porous. 
Each  of  these  changes  react  upon  the  water-holding  capacity  and 
the  capillarity  of  the  soil,  greatly  altering  the  aeration  of  the  soil 
and  with  the  aeration  the  temperature. 

The  biological  changes  which  the  manure  produces  in  the  soil, 
especially  when  small  quantities  are  added,  may  be  even  more  far- 
reaching  than  either  the  chemical  or  physical  changes  which  it 
produces.  Every  pound  of  manure  carries  with  it  to  the  soil  millions 
of  bacteria.  Many  of  these  will  find  the  new  conditions  unsuited  for 
their  growth,  but  some  will  continue  to  multiply,  and  in  so  doing 
not  only  will  decompose  the  constituents  of  the  manure  but  also 
will  greatly  alter  other  organic  and  inorganic  substances  of  the  soil. 
The  bacterial  content  of  the  soil  is,  therefore,  changed  both  quanti- 
tatively and  qualitatively.  There  are  added  with  the  manure  many 
new  species;  the  changed  physical  and  chemical  conditions  of  the 
soil  due  to  the  manure  will  greatly  modify  those  already  present, 
for  the  microflora  and  microfauna  originally  present  in  the  soil  were 
due  to  specific  soil  properties. 

This  changed  flora  and  fauna  will  in  turn  change  the  chemical 
and  physical  properties  of  the  soil  still  more.  Acids  are  generated, 
which  react  with  insoluble  constituents,  rendering  them  soluble. 
Gases  are  formed,  which  change  the  air  within  the  soil;  in  these 
reactions  heat  is  generated,  thus  changing  the  temperature  of  the 
soil.  The  metabolism  of  the  bacterial  cell  requires  nutritive  sub- 
stances, among  which  are  water  and  the  elements  essential  to  plant 
growth.  Some  soluble  constituents  will  be  changed  to  insoluble  and 


151 

some  inorganic  to  organic.  All  of  these  changes  are  reflected  in  the 
crop  yield. 

Number.— That  the  addition  of  manure  to  a  soil  increases  the 
number  of  bacteria  has  been  shown  by  Remy  and  Fischer. 

Caron  found  that  the  number  of  bacteria  present  depends  not 
only  upon  the  manure  added  but  upon  the  cultural  methods  and  the 
crop  grown  on  the  soil*.  Fabricius  and  von  Feilitzen  found  that 
bacteria  increased  in  the  soil  on  the  addition  of  manure  and  that  a 
direct  relationship  existed  between  the  temperature  of  a  soil  and 
the  number  of  bacteria  found  in  it.  That  the  temperature  of  the 
soil  is  influenced  by  the  addition  of  manure  is  shown  by  Wagner  who 
observed  that  manure  increased  the  temperature  of  soil  from  1  to 
2.8°  C.,  depending  on  the  kind  and  condition  of  manure  added. 
Troop  noted  an  average  increase  of  5°  in  temperature  of  soil  receiving 
25  tons  an  acre  of  manure  over  an  unmanured  soil.  Petit,  however, 
claimed  that,  while  there  was  at  first  an  increase  in  the  temperature 
of  manured  soils,  later  it  became  lower  than  the  unmanured.  Stigell 
concluded  that  bacteria  under  favorable  conditions  for  de  relopment 
retarded  the  conduction  of  heat  in  soils  and  thereby  reduced  the 
temperature  changes  due  to  the  variation  in  the  outside  temperature. 
This,  in  a  way,  might  neutralize  the  effect  of  manure,  for  Hecker 
found  that  although  the  temperature  of  soil  to  which  well-rotted 
manure  had  been  added  was  higher  than  adjacent  unmanured  soil 
during  the  day,  the  opposite  was  true  during  the  night.  Grazia 
stated  that  manures  greatly  increase  the  temperature  of  the  soil. 
King  found  that  a  definite  increase  in  bacterial  activity  occurred 
with  increased  temperature,  but  that  an  excessive  moisture  content 
greatly  reduced  the  number  of  bacteria  in  a  soil.  Engberding 
claimed  that  manure  increased  the  number  of  bacteria  in  a  soil,  but 
he  considered  that  the  moisture  content  had  a  greater  influence  on 
numbers  than  did  temperature.  That  the  moisture  content  greatly 
influenced  bacterial  activity  was  shown  by  Deherain  and  Demoussy, 
who  found  that  the  bacterial  action  of  a  soil  was  at  its  maximum 
when  a  rich  soil  contained  17  per  cent,  of  water,  but  that  it  decreased 
if  the  proportion  of  water  fell  to  10  per  cent,  or  rose  to  25  per  cent. 
With  soils  less  rich  in  humus  a  somewhat  higher  proportion  of  water 
was  necessary  to  retard  oxidation  to  any  marked  degree.  In  a 
manured  soil  the  coarse  manure  tended  to  cause  the  surface  soil  to 
dry  out,  while  fine  manure  prevented  evaporation.  King  observed 
that  manured  land  contained  more  moisture  throughout  the  year 
than  unmanured,  and  this  was  reflected  in  both  a  greater  number 
of  bacteria  and  in  a  larger  crop.  The  bacteria  themselves  may 
play  a  small  part  in  this  difference  in  moisture  content,  as  was 
shown  by  Stigell,  who  found  that  bacteria  decreased  the  speed  of 
evaporation  of  water  from  Petri  dishes.  Hiltner  and  Stormer's 
results  indicate  that  the  addition  of  manure  to  a  soil  brought  about 


152 


INFLUENCE  OF  MANURE  ON  THE  SOIL 


a  marked  change  in  the  number  of  bacteria.  The  temperature, 
cultural  methods,  and  crop  had  an  influence,  but  it  was  not  nearly 
so  pronounced  as  that  produced  by  the  manure,  as  is  seen  from  the 
following  in  which  is  given  the  number,  stated  as  millions  per  gram, 
of  bacteria  found  in  manured  and  unmanured  soils  at  different 
seasons. 


1901. 

1902. 

10  May. 

27  Aug. 

18  Oct. 

IFeb. 

12  June. 

18  Aug. 

Cropped  land,  grass  and  clover    . 
Cultivated  fallow,  unmanured 
Cultivated  fallow,  manured    .      . 

8.3 
8.0 
11.0 

3.2 
4.2 
10.5 

6.4 
4.0 
11.0 

6.6 
4.1 
9.3 

8.1 
5.7 
7.2 

4.9 
4.1 

8.4 

Brown,  in  a  study  of  the  influence  of  manure  on  the  bacterial 
activities  of  a  loam  soil,  found  that  applications  of  manure  up  to  16 
tons  an  acre  increased  the  number  of  bacteria  and  also  the  ammoni- 
fying and  nitrifying  powers  of  the  soil.  The  greatest  increase  in  the 
processes  was  brought  about  by  small  applications  of  manure,  8  to  12 
tons  to  the  acre.  He  observed  a  close  relationship  between  the 
ammonifying  powers  of  the  soil,  the  bacterial  content,  and  the  crop 
produced  on  the  soil. 

Temple  stated  that  the  addition  to  a  soil  of  10  tons  an  acre  of  cow 
manure  greatly  increased  the  number  of  bacteria  in  the  soil,  but 
that  a  greater  increase  occurred  when  a  sterilized  manure  was  applied. 
This,  however, -is  not  in  keeping  with  the  results  obtained  by  other 
investigators.  Hellstrom  concluded  that  manures  possessed  a 
fertilizing  effect  aside  from  the  quantities  of  fertilizer  constituents 
contained  within  them;  and  this,  he  maintained  is  due  to  their  great 
bacterial  content.  Stoklasa  found  that  manure  increased  the  bac- 
terial content  and  activity  of  a  soil,  the  increase  being  greater  with 
small,  frequent  applications  of  manure  than  with  large  applications 
made  at  longer  intervals.  Moreover,  Lipman  and  others  observed 
that  the  bacteria  conveyed  to  soil  in  small  quantities  of  manure 
were  valuable  in  bringing  about  a  more  rapid  decomposition  of  a 
green-manure  crop.  Briscoe  said  that  a  direct  relationship  existed 
between  the  organic  matter  added  to  a  soil  and  the  bacterial  count, 
and  that  a  light  dressing  of  manure  with  green  manure  produced  a 
marked  increase  upon  both  the  yield  of  the  crop  and  number  of 
bacteria.  Bacterial  cultures  added  with  the  green  manure  gave  just 
as  pronounced  an  effect  as  did  the  stable  manure.  Lemmermann 
and  Einecke,  however,  obtained  no  increase  on  adding  stable  manure 
with  green  manure.  This  may  be  due  to  the  different  kind  of  manure 
used,  for  Emmerich  and  others  maintained  that  a  more  favorable 
effect  was  obtained  from  the  use  of  well-rotted  manure  than  from  the 


LOSS  OF  NITRATES  153 

use  of  fresh  manure.  This,  they  maintained,  was  due  to  the  pro- 
duction in  the  latter  of  formic,  acetic,  and  butyric  acid,  indol,  skatol, 
and  hydrogen  sulphid,  which  are  toxic  to  the  plant.  Under  some 
conditions,  the  large  quantities  of  carbon  dioxid  liberated  from  the 
rapidly  decomposing  fresh  manure  may  be  valuable  in  rendering  the 
plant-food  soluble.  Bornemann  found  that  soil  constantly  supplied 
witn  carbon  dioxid  through  a  pipe  buried  in  the  ground  gave  an 
increase  in  yield  of  12.2  per  cent,  over  the  crop  grown  on  untreated 
soil.  Wollny  has  shown  that  manure  greatly  increased  the  carbon- 
dioxid  production  in  a  soil. 

Ammonification  and  Nitrification.— Moll  considered  that  the  season 
of  the  year,  and  not  the  kind  of  fertilizer  used,  nor  even  the  weather 
conditions,  is  the  principal  factor  in  determining  the  extent  of  pep- 
tone decomposition,  nitrification,  and  nitrogen  fixation  of  a  soil. 
According  to  Wohltmann,  Fischer,  and  Schneider,  ammonification, 
nitrification,  and  nitrogen  fixation  were  all  more  or  less  increased 
by  the  application  of  manure.  Heinze  found  that  manure  was 
especially  beneficial  to  the  nitrifying  organisms.  Warington  reports 
that  much  more  nitric  nitrogen  was  found  in  the  soil  of  plots  which 
had  received  annually  for  thirty-eight  years  a  dressing  of  14  tons  of 
manure  to  the  acre  than  in  any  of  the  other  manured  or  unmanured 
plots.  Stevens  found  that  .nitrification  was  much  more  active  in 
manured  than  in  unmanured  soil,  but  Frankfurt  and  Duschechkin 
observed  an  increase  in  nitrification  only  on  those  manured  plots  on 
which  the  yield  had  increased.  Welbel  has  shown  that  the  chief 
factors  controlling  nitrification  in  fallow  soil  were  the  humus  and 
the  humus-nitrogen  content,  the  nitrification  having  increased 
directly  with  the  humus.  He  noted,  however,  a  certain  amount  of 
denitrification  at  first,  but  later  in  the  summer  nitrification  became 
more  rapid  on  the  manured  than  on  the  unmanured  soil,  the  effect 
of  the  manure  being  still  perceptible  after  four  years.  Some  investi- 
gators have  reported  a  reduction  of  nitrates,  but  the  quantity  of 
manure  applied  was  excessive,  or  else  of  a  very  coarse  nature,  or  the 
soil  poorly  aerated.  Barthel  found  that  nitrification  did  not  take 
place  in  the  presence  of  soluble  organic  matter,  but  he  considered 
it  unlikely  that  sufficient  quantities  of  soluble  organic  constituents 
occurred  in  normal  agricultural  soils  to  interfere  greatly  with  nitri- 
fication. Niklewski  maintained  that  nitrification  occurred  in  solid 
stable  manure  when  there  was  not  much  liquid  present.  He  stated 
that  on  the  first  day  some  nitrite  bacteria  were  present  and  at  the 
end  of  four  weeks  there  were  10,000  in  each  gram.  Associated  with 
these  were  nitrate  bacteria  which  were  identical  with  those  isolated 
by  Winogradsky.  Millard,  however,  was  unable  to  find  many  nitrify- 
ing bacteria  in  manure. 

Loss  of  Nitrates.— Many  of  the  cases  in  which  individuals  have 
reported  a  disappearance  of  nitrates  in  soil  are  due  to  synthetic 


154  INFLUENCE  OF  MANURE  ON  THE  SOIL 

reactions,  the  nitrates  being  built  up  into  complex  proteins.  For 
Gerlach  and  Vogel  have  shown  that  there  are  several  varieties  of 
bacteria  in  the  soil  which  have  the  power  of  converting  ammonia, 
nitrites,  and  nitrates  into  insoluble  proteins. 

It  is  evident  from  the  literature  cited  that  there  is  a  wide  variation 
in  the  ideas  held  concerning  the  influence  of  manure  upon  the 
bacterial  flora  of  the  soil.  This  is  due  to  a  number  of  factors,  chief 
among  which  are:  (1)  variation  in  the  physical  and  chemical  com- 
position of  the  soil;  (2)  a  great  variation  in  the  composition  of  the 
manure  used;  and  (3)  the  manure  added  may  have  influenced  either 
beneficially  or  injuriously  the  water  content  of  the  soil.  The  results 
noted  may  have  been  due  to  the  moisture  factor  and  not  to  the 
manure.  An  experiment  was  conducted  at  the  Utah  Experiment 
Station  both  in  pots  and  under  field  conditions.  Each  ton  of  the 
manure  added  was  partly  rotted  and  contained  in  one  ton  734  pounds 
of  dry  matter,  3.04  pounds  of  phosphorus,  13.7  pounds  of  potassium, 
and  16.08  pounds  of  nitrogen. 

The  conclusions  reached  follow:  A  calcareous  soil  kept  in  pots 
with  varying  amounts  of  manure  and  different  percentages  of  moist- 
ure gave  on  bacteriological  analysis  at  the  end  of  four  months  the 
following  results :  The  temperatures  of  the  manured  and  unmanured 
were  practically  the  same  for  the  period,  but  the  temperature  of  the 
soil  with  12.5  per  cent,  of  water  was  1°  C.  higher  than  soils  with 
22.5  per  cent,  of  water.  The  greatest  number  of  organisms  devel- 
oped on  synthetic  media  from  the  soils  receiving  the  greatest  quan- 
tity, 25  tons,  of  manure.  There  were  more  colonies  developed  from 
the  soil  receiving  12.5  per  cent,  of  water  than  from  any  of  the  other 
soils  receiving  higher  quantities  of  water. 

The  nitrifying  powers  of  the  soil  increased  as  the  manure  and  water 
applied  increased  up  to  25  tons  of  manure  and  22.5  per  cent,  of 
water. 

The  nitrogen-fixing  powers  of  the  soil  were  greatest  in  those  pots 
receiving  manure  at  the  rate  of  10  tons  an  acre.  Increasing  the 
water  above  12.5  per  cent,  but  not  above  22.5  per  cent,  slightly 
increased  the  nitrogen-fixing  powers  of  the  soil.  Nothing  in  the 
results  indicated  that  the  application  of  manure  up  to  25  tons  an 
acre  and  of  water  up  to  22.5  per  cent,  caused  denitrification  in  the 
soil. 

Bacteriological  analyses  of  fallow  field  soil  receiving  no  manure, 
5  tons,  and  15  tons  an  acre  and  receiving  no  water,  5  inches,  10  inches, 
20  inches,  30  inches,  and  40  inches  of  irrigation  water,  indicated  that 
the  maximum  number  of  bacteria  were  obtained  from  the  soil 
receiving  15  tons  of  manure.  The  application  of  irrigation  water 
up  to  20  inches  increased  the  bacterial  count,  the  increase  being 
most  noticeable  in  the  soil  receiving  the  greatest  quantity  of  manure. 

If  the  ammonifying  power  of  the  unmanured  soils  is  considered 


LOSS  OF  NITRATED 


155 


as  100  per  cent,  and  that  of  the  unirrigated  as  100  per  cent.,  the 
manured  and  irrigated  soils  then  become  with  5  tons  of  manure,  147 
per  cent.;  with  15  tons  of  manure,  188  per  cent.;  5  inches  of  water, 
106  per  cent.;  10  inches  of  water,  117  per  cent.;  20  inches  of  water, 


EXPLANATION 

i 

3060 

1         PER  CENT  OF  GRAIN 

H         PER  CENT  OF  STOVER 

DH          PER  CENT  OF  BACTERIA 

500 

H          PER  CENT  OF  BACTERIA    (FALLOW) 

0          PER  CENT  OF  AMMONIA 

H          PER  CENT  OF  AMMON  A   (FALLOW) 

$c 

480 

gj         PER  CENT  OF  N  TRIC  NITROGEN 

58 

§|         PER  CENT  OF  N  TR  C 

NITROGEN  (FALLOW) 

1 

900 

\  Jx 

180 

u 

M 

^  w 

3 

IdO 

-- 

a  N 

1 

|| 

120 

= 

i   N, 

V  T      N 

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/  | 

'- 
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llliiKM      i 

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.1 

H   Ba^KeS          E 

ll  ^ 

o 

100 

j-jl  jii::f>:;:'~        \ 

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I 

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. 

60 

Afl 

|J  it   j 

; 
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5  TONS 
OF  MANURE 


15  TONS 
OF  MAN.URE 


FIG.  23.— Diagram  showing  the  influence  of  manure  on  the  yield  and  bacterial  activi- 
ties of  a  soil.    The  unmanured  soil  is  expressed  as  100  per  cent. 

108  per  cent.  Large  quantities  of  irrigation  water  produced  the 
greatest  depressing  effect  in  the  presence  of  15  tons  of  manure  to  the 
acre. 

Fewer  organisms  develop  on  synthetic  agar  from  a  cropped  than 
from  a  fallow  soil.     The  application  of  manure  to  a  cropped  soil 


156  INFLUENCE  OF  MANURE  ON  THE  SOIL 

increases  the  bacterial  count  of  the  soil.  The  greatest  number  of 
organisms  developed  from  the  soil  receiving  10  inches  of  irrigation 
water. 

The  ammonifying  power  of  the  cropped  soils  was  slightly  lower 
than  similarly  treated  fallow  soils.  The  application  of  5  and  15 
tons  of  manure  on  each  acre  increases  the  ammonifying  power  of  the 
soil.  The  application  of  irrigation  water  up  to  30  inches  increases 
the  ammonifying  power  of  the  soil.  The  greatest  increase  resulted 
in  those  soils  receiving  15  tons  of  manure  to  the  acre.  The  applica- 
tion of  40  inches  of  irrigation  water  to  corn  land,  especially  to  that 
receiving  15  tons  of  manure  an  acre,  depresses  the  ammonifying 
power  of  the  soil. 

The  nitrifying  power  of  fallow  soil  was  higher  than  similarly 
treated  cropped  soils.  The  application  of  manure  to  a  cropped  soil 
greatly  increases  the  rate  of  nitrification.  The  application  of  irriga- 
tion water  up  to  30  inches,  especially  to  a  soil  receiving  15  tons  of 
manure,  greatly  increases  its  nitrifying  power. 

Green  manures  are  rapidly  taking  the  place  of  bare  fallows  in  all 
of  the  better  systems  of  agriculture  where  the  rainfall  will  permit  of 
the  practice.  The  practice  of  green-manuring  consists,  essentially, 
of  the  turning  under  of  green  crops  for  the  benefit  of  succeeding 
crops.  In  addition  to  the  various  legumes,  which  are  preferred  on 
account  of  their  ability  to  take  nitrogen  direct  from  the  air,  crops 
like  rye,  wheat,  oats,  buckwheat,  mustard,  rape  and  even  turnips 
have  been  used  more  or  less  extensively  as  green  manures. 

The  plowing  under  of  green  manure  produces  either  a  beneficial 
or  injurious  effect,  depending  upon  the  nature  of  the  soil  to  which  it 
is  applied,  the  kind  of  manure  added,  and  the  season  of  the  year 
when  applied.  Some  of  the  beneficial  influences  noted  are: 

1.  They  carry  to  the  soil  large  quantities  of  organic  matter  which 
on  decaying  yield  humus,  and  this  in  turn  changes  materially  the 
physical  and  chemical  composition  of  the  soil.  Schultz  started  in 
1855  on  an  extremely  poor,  coarse-grained,  sandy  soil,  and  gradually 
improved  it  by  the  use  of  lime,  phosphoric  acid,  and  potash  in  con- 
nection with  such  green-manuring  crops  as  lupines,  serradella,  and 
field-peas,  until  he  could  produce  three  hundred  to  four  hundred 
bushels  of  potatoes  to  the  acre.  Neale  noted  a  marked  gain  in  the 
yield  of  corn  when  crimson  clover  was  used  as  a  green  manure.  He 
believed  the  nitrogen  thus  applied  to  be  much  more  economical  than 
when  nitrate  of  soda  is  used.  For  sandy  soils  the  results  of  Delwiche 
would  indicate  cowpeas,  hairy  vetch,  soy  beans,  and  crimson  clover 
to  be  best,  and  Pfeiffer's  results  indicate  that  it  is  the  open,  sandy 
soils  which  give  the  best  results  with  green  manures.  The  actual 
effect  produced,  however,  varies  with  the  time  of  application. 
Bassler  recommends  that  where  lupines,  serradella,  crimson  clover, 
and  hairy  vetch  are  used  for  green  manure  on  sandy  soils  they  should 


LOSS  OF  NITRATES  157 

be  turned  under  as  late  as  possible  in  the  life  of  the  plants  and  not 
in  the  hot  summer  when  the  plants  are  green.  This  agrees  with  the 
findings  of  Causemann  who  recommends  that  they  be  turned  under 
late  in  September.  It  has  been  observed  by  Bredemann  that  the 
addition  of  organic  matter,  such  as  hay  and  sugar,  produces  a  harm- 
ful effect  the  first  year  and  a  beneficial  effect  the  two  following  years. 

2.  Green  manures  change  the  number  and  kind  of  organisms 
occurring  in  the  soil.  Hill  found  the  total  number  of  bacteria  in 
soils  treated  with  green  manures  to  be  much  greater  than  in  those 
receiving  no  green-manure  treatment.  Legumes  gave  in  most  cases 
a  greater  increase  than  non-legumes.  Miintz  considers  the  value  of 
green  manures  proportional  to  the  rapidity  with  which  their  nitrogen 
is  converted  into  nitric  nitrogen.  Heinze,  on  contrasting  stall  and 
green  manures,  found  that  the  latter  carried  but  few  organisms  which 
would  break  down  the  insoluble  material.  The  decomposition  of 
green  manure  was  found  to  be  due  to  dust  organisms  and  to  organ- 
isms found  in  soil.  For  this  reason  the  number  and 'kind  of  organisms 
in  a  given  soil  determine  in  a  great  measure  the  influence  of  green 
manures  on  succeeding  crops.  Decomposition  is  rapid  in  an  open, 
sandy  soil  rich  in  bacteria  and  relatively  slow  in  a  soil  poor  in  bacteria. 
In  an  open,  sandy  soil  the  nitrogen  of  the  green  manure  may  pass 
over  into  nitrates  and  be  washed  out,  whereas  in  a  heavier  soil  the 
nitrogen  becomes  available  more  slowly  and  is  not  washed  out  so 
rapidly.  For  this  reason  in  heavy  soils  green  manures  often  give  the 
best  results  the  second  year. 

Koch  thinks  the  good  effects  produced  when  green  manure  is 
added  .to  stable  manure  may  be  due  to  an  increased  nitrogen  fixation 
by  Azotobacter,  the  organism  using  the  cellulose  of  the  manure  as  a 
source  of  energy;  whereas  Heinze  considers  that  the  results  which  he 
obtained  from  studying  the  action  of  carbon  bisulphid  on  soils  may 
help  us  to  understand  the  peculiar  effects  produced  at  times  by  the 
turning  under  of  mustard,  buckwheat,  rye,  and  other  non-leguminous 
crops.  It  has  been  noted  repeatedly  that  these  crops  when  plowed 
under  in  a  green  state  lead  to  a  better  growth  of  the  following  cereal 
or  root  crops  on  nitrogen-poor  soils.  As  Heinze  points  out,  there 
may  have  been  more  or  less  justification  for  this  belief,  so  far  as  the 
indirect  influence  of  mustard  is  concerned.  It  would  seem  at  times 
that  the  action  of  mustard  is  not  unlike  that  of  carbon  bisulphid  in 
affecting  the  bacterial  flora  of  the  soil,  and  it  really  appears  from 
facts  already  known  that  the  green  mustard  substance  in  the  soil 
retards  the  development  of  the  acid-forming  species  and  encourages 
the  growth  of  the  nitrogen-fixing  Azotobacter  species.  Heinze, 
therefore,  thinks  that  further  study  may  enable  us  to  make  extensive 
use  of  mustard  as  an  indirect  source  of  combined  nitrogen,  and  tries 
to  find  theoretical  support  for  this  belief  in  the  fact  that  allyl 
isothiocyanid  mustard  oil,  C3H5  —  N  =  C  =  S,  which  is  a  constit- 


158  INFLUENCE  OF  MANURE  ON  THE  SOIL 

uent  of  the  mustard  plant,  may  be  regarded  as  a  derivative  of 
carbon  bisulphid  and  probably  would  have  a  similar  influence  upon 
the  soil  microflora. 

3.  The  growing  of  a  crop  during  the  season  of  the  year  when  the 
heavy  rains  would  wash  much  of  the  nitric  nitrogen  beyond  the 
roots  of  the  plants  prevents  this  loss,  for  the  nitrogen  is  stored  in 
the  body  of  the  plants  and  is  later  liberated  by  decomposition  for 
succeeding  plants. 

4.  The  growth  of  the  legumes  may  actually  lead  to  an  increase  in 
the  nitrogen  of  the  soil.    The  method,  extent,  and  conditions  under 
which  this  occurs  is  considered  in  detail  in  later  chapters. 

The  results,  however,  following  the  use  of  green  manures  are  not 
uniformly  beneficial,  for  the  following  ill  effects  have  at  times 
followed  its  use: 

1.  The  physical  condition  of  the  soil  may  be  injured.    It  becomes 
too  loose  and  open,  decomposition  being  thereby  decreased  and 
leaching  increased.    This  was  probably  the  reason  why  Brown  did 
not  always  find  an  increase  in  the  bacterial  activities  following  the 
application  of  green  manure.    This  is  also  true  with  regard  to  the 
work  reported  by  Laurent. 

2.  Engberding,  studying  the  effect  of  straw  and  sugar  upon  the 
number  of  bacteria  in  the  soil,  found  at  first  an  increase,  followed  by 
a  decrease.   The  ammonifying  and  nitrogen-fixing  groups  of  bacteria 
showed  an  increase,  but  the  nitrifying  group  was  retarded. 

Fischer,  in  his  paper  on  the  changes  undergone  by  nitrogen  in 
sandy  and  clayey  soils,  offers  an  explanation  for  the  loss  of  nitrates 
in  a  soil  to  which  carbohydrates  have  been  added  in  the  following 
reaction : 

24  NaNOs  +  5  C6Hi2O6  =  24  NaOH  +  12  N2  +  30  CO2  +  18  H2O 

The  oxygen  of  the  nitrate  is  used  by  the  ferments  for  the  oxidation 
of  the  carbohydrate  and  the  nitrogen  is  liberated  as  a  gas. 

Frankfurt  and  Duschechkin  state  that  green  manure  under  field 
conditions  caused  a  diminution  of  the  nitrate  content.  Both  legumes 
showed  this  effect,  but  they  consider  it  as  due  to  the  action  of  the 
manure  upon  the  soil  moisture. 

Stevens  and  Withers  give  two  reasons  why  the  activity  of  nitrify- 
ing organisms,  in  pure  culture  under  laboratory  conditions,  cannot  be 
compared  with  their  activities  under  conditions  in  the  field :  (1)  "  In 
mixed  culture  their  symbiotic  and  physiologic  relations  are  so 
different  from  those  obtained  in  pure  cultures  that  their  metabolic 
processes  are  with  difficulty  expressed;"  (2)  "the  presence  of  large 
amounts  of  solid  matter,  sand,  or  earth,  in  contact  with  the  liquid 
medium,  so  alters  its  relation  to  the  nitrifying  organisms  that  their 
physiologic  activities  and  metabolic  products  are  different."  These 
authors  say  in  conclusion;  "In  the  light  of  the  facts,  $&  forth,  the 


LOSS  OF  NITRATES  159 

direct  application  of  Winogradsky's  conclusions  to  the  field  must  be 
abandoned  and  with  them  any  practices  based  upon  them,  and  the 
activities  of  these  soil  bacteria  must,  in  the  future,  be  studied  more 
largely  under  their  natural  environments." 

Lipman,  commenting  on  these  earlier  views,  believes  that  "the 
exact  relation  of  organic  matter  in  the  soil  to  the  activities  of  nitrify- 
ing bacteria  is  but  beginning  to  be  properly  understood.  Earlier 
observation  made  it  manifest  that  heavy  applications  of  animal 
manures,  or  green  manure,  may  not  only  retard  nitrification  but 
may  actually  cause  the  disappearance  of  a  part  or  of  all  of  the  nitrate 
in  the  soil.  Subsequent  experiments  by  Winogradsky  and  Omelianski 
showed  that  in  pure  cultures  the  presence  of  even  slight  amounts 
of  soluble  organic  matter  may  depress  or  even  suppress  the  develop- 
ment of  the  nitrifying  bacteria.  It  was,  therefore,  concluded  by 
these  authors  that  relatively  small  amounts  of  soluble  organic 
matter  may  inhibit  nitrification.  These  conclusions,  based  on  the 
study  of  liquid  cultures  only,  were  given  a  very  broad  application 
by  many  writers  on  agricultural  topics.  More  recent  experiments 
make  it  certain,  however,  that  in  the  soil  itself  small  amounts  of 
soluble  matter,  for  example,  dextrose,  are  not  only  harmless  but 
may  really  stimulate  nitrification.  It  was  shown,  likewise,  that 
humus  and  extracts  of  humus  may,  under  suitable  conditions,  stimu- 
late nitrification  to  a  very  striking  extent." 

REFERENCE. 

Greaves,  J.  E.,  and  Carter,  E.  G.:  Influence  of  Barnyard  Manure  and  Water  upon 
the  Bacterial  Activities  of  the  Soil  (Journal  Agricultural  Research,  1916,  vi,  889-926). 


CHAPTER  XVI. 
THE  SOIL  FLORA. 

Two  methods  are  in  general  use  for  the  quantitative  determina- 
tion of  bacteria  in  the  soil:  the  Koch  gelatin-plate  method  and  the 
Hiltner  and  Stormer  dilution  method. 

Koch  Gelatin-plate  Method.— The  determination  by  the  gelatin- 
plate  method  is  made  as  follows :  Samples  of  soil  are  carefully  mixed, 
usually  by  shaking,  and  100  grams  of  the  mixed  soil  weighed  on  sterile 
paper  or  watch-glasses  into  200  c.c.  of  sterile  water.  This  is  shaken, 
for  one  minute.  Ten  c.c.  of  this  is  transferred  to  90  c.c.  of  sterile 
water.  This  is  continued  until  the  proper  dilution  is  obtained, 
usually  1  to  20,000  and  1  to  200,000,  then  they  are  plated  on  gelatin 
or  some  other  solid  media.  The  number  of  colonies  developing  is 
counted  at  the  end  of  four  or  seven  days.  The  longer  period  is  pref- 
erable for  many  of  the  important  soil  organisms  are  missed  in  the 
shorter  incubation  period. 

Hiltner  and  Stormer  Dilution  Method.— The  Hiltner  and  Stormer 
method  uses  solutions  prepared  to  favor  in  each  case  the  develop- 
ment of  one  of  the  various  groups  of  bacteria  (ammonifying,  nitrify- 
ing, nitrogen-fixing,  etc.).  The  soil  is  inoculated  into  the  appro- 
priate media  in  constantly  decreasing  quantities  usually  100,  10,  1, 
0.1,  0.01,  2,  and  0.001  mg.  of  soil.  The  material  is  incubated  and 
after  the  appropriate  time  the  gain  in  ammonia,  nitrites,  nitrates,  etc. 
determined.  By  this  procedure  a  point  is  finally  reached  where  the 
small  quantity  of  soil  employed  contains  none  of  the  specific  organ- 
isms, and  hence  fails  to  give  rise  to  the  specific  physiological  reaction. 
By  this  method  a  fairly  accurate  estimation  may  be  made  of  the 
number  of  different  microorganisms  in  the  soil. 

Lohnis  clearly  demonstrated  that  the  Hiltner  and  Stormer  method 
is  more  exact  than  is  the  gelatin-plate  method.  He  found  that 
a  soil  which  yielded  1,270,000  bacteria  per  gram  by  the  gelatin- 
plate  method,  by  the  Hiltner  and  Stormer  method  yielded  3,750,000 
peptone-decomposing  bacteria,  50,000  urea-decomposing  bacteria, 
50,000  denitrifying  bacteria,  7000  nitrifying  bacteria,  and  25 
nitrogen-fixing  bacteria,  making  a  total  of  3,857,025,  which  is  over 
three  times  the  number  obtained  by  the  plate  method. 

Defects  of  Plate  Method.^- The  number  of  organisms  found  in  soil 
by  this  method  varies  with  the  media  used  and  the  period  elapsing 
between  plating  and  counting.  But  even  under  the  most  favorable 


NUMBER  OF  BACTERIA  IN  SOIL  161 

conditions,  the  numbers  are  far  below  the  number  actually  occurring 
in  the  soil.    Some  of  the  reasons  for  this  are  as  follows : 

1.  Even  many  of  the  peptone-decomposing  bacteria  fail  to  grow 
on  the  gelatin  plates.    This^may  be  due  to  overcrowding  or  to  the 
sudden  change  of  conditions  and  the  resulting  osmotic  disturbances. 

2.  The  nitrifiers  do  not  grow  on  the  ordinary  organic  laboratory 
media;  moreover,  they  have  not  been  found  in  soil  in  sufficient 
numbers  to  occur  on  plates  dilute  enough  to  show  the  more  abundant 
organisms.     The  nitrogen-fixing  organisms— both  symbiotic  and 
non-symbiotic— usually  occur  in  soil  in  too  small  a  number  to  be 
noted  by  the  ordinary  plate  method. 

3.  The  strict  anaerobes  which  occur  in  soils  in  vast  numbers  do 
not  grow  on  the  gelatin  plate  under  the  ordinary  conditions  of 
aerobic  culture. 

4.  No  medium  yet  devised  resembles  the  soil  in  composition  and 
structure,  and  hence  the  plate  does  not  necessarily  reflect  the  flora 
active  in  the  soil.    Moreover,  it  is  impossible  to  tell  which  of  the 
forms  developing  on  the  plate  are  active  and  which  are  spores  in  the 
soil. 

A  third  method  for  the  determination  of  the  bacteria  in  the  soil 
is  the  direct  microscopic  count.  This  method,  however,  has  not 
been  used  sufficiently  as  yet  to  permit  a  conclusion  as  to  its  relative 
value. 

Value  of  Bacterial  Counts.— The  methods  for  •  determining  the 
number  of  bacteria  in  soil  are  admittedly  faulty;  yet  they  have  the 
advantage  of  showing  whether  the  number  is  high  or  low  and  whether 
they  are  increasing  or  decreasing.  The  counts  show  fairly  accurately 
whether  any  given  treatment  of  the  soil  has  raised  or  lowered  the 
number  of  bacteria  in  the  soil.  But  numbers  alone  furnish  only 
meager  information,  for,  as  pointed  out  by  Remy,  the  number  of 
colonies  of  aerobic  soil  bacteria  appearing  on  plates  show  no  direct 
relationship  to  the  ammonifying,  nitrifying,  or  denitrifying  powers 
of  the  corresponding  soil.  Lohnis  is  even  more  emphatic  than  Remy 
in  designating  mere  quantitative  methods  as  untrustworthy.  He 
points  out  that  it  is  quite  possible  that  two  million  very  efficient 
ammonia-producing  bacteria  present  in  1  gram  of  soil  will  accomplish 
more  work  than  five  million  less  efficient  ones  will  in  another  soil. 

This  same  principle  is  brought  out  by  Chester  when  he  states 
that  a  soil  may  be  low  in  the  total  number  of  bacteria,  but  contain 
such  a  bacterial  flora,  or  combination  of  bacterial  species,  which 
are  known  to  be  favorable  to  the  rapid  digestion  of  plant-food,  as 
to  give  it  what  might  be  termed  a  high  bacterial  potential.  In  other 
words,  he  holds  that  we  should  consider  not  alone  numbers  but  also 
physiological  efficiency. 

Number  of  Bacteria  in  Soil.— The  number  of  bacteria,  as  deter- 
mined by  the  plate  method,  in  good  arable  soil  well  supplied  with 
11 


162  THE  SOIL  FLORA 

organic  matter  usually  ranges  from  three  to  forty  million.  Tight, 
non-porous  soils,  as  well  as  alkali  and  soils  low  in  humus  and 
moisture,  yield  low  bacterial  counts. 

The  number  of  microorganisms  in  light,  sandy,  very  tight  clay, 
desert  and  forest  soils  is  usually  much  smaller,  other  things  being 
equal,  than  in  normal  cultivated  soils.  Cultivation  of  soils  tends 
to  increase  greatly  the  number  of  bacteria.  The  average  of  several 
hundred  determinations  made  on  cultivated  soil  of  the  arid  regions 
gave  a  bacterial  count  of  4,452,000,  whereas  the  average  of  a  similar 
number  of  samples  taken  from  adjoining  virgin  soils  was  2,270,000. 

Factors  Influencing  Number.— Optimum  moisture,  organic  matter 
and  aeration  tend  to  increase  the  number  as  does  also  the  addition 
of  sugar  and  certain  antiseptics : 

Bacterial  number  after  50  days. 

Soil  treated  with  antiseptics, 
Control  soil.  millions  per  gram. 

Cane  sugar  (p.  25  per  cent.)    ...  21  51 

Amyl  alcohol  (0.1  per  cent.)    ...  30  85 

Phenol  (m/200  per  kilo)      ....  27  101 

Hydroquinone  (m/200  per  kilo)  16  55 

There  is  a  direct  relation  between  the  number  of  bacteria  found 
in  the  soil  and  the  quantity  of  organic  manure  added.  This  is 
illustrated  in  results  obtained  by  the  author  and  given  in  tabular 
form  below.  The  unmanured  soil  is  taken  as  100  per  cent. 

Treatment.  Fallow  soil.  Cropped  soil. 

No  manure .      100  100 

5  tons  of  manure 144  123 

15  tons  of  manure 177  129 

The  aeration  of  the  soil  often  increases  manyfold  the  number  of 
bacteria,  and  according  to  Conn  the  greatest  numerical  increase 
occurs  in  the  group  of  non-spore-forming  bacteria. 

The  number  of  bacteria  found  in  the  soil  varies  considerably 
with  the  season  of  the  year.  A  very  interesting  phenomenon,  noted 
by  Conn  and  confirmed  by  Brown,  was  that  the  number  of  bacteria 
in  soil  increase  on  freezing.  This  fact  is  illustrated  in  the  following 
table  from  the  work  of  Conn.  In  it  are  listed  a  series  of  gelatin- 
plate  counts  made  from  a  single  soil  plate  at  intervals  throughout 
the  course  of  three  years. 

During  the  three  years  over  which  the  sampling  was  conducted 
the  plate  count  of  non-spore-formers  varied  from  5,000,000  to 
44,000,000  per  gram,  whereas  the  spore-forming  bacteria  and 
Actinomycetes  varied  only  from  3,200,000  to  10,500,000.  The 
increase  which  occurs  during  the  winter  is  in  the  slow-growing 
bacteria  and  not  in  those  which  liquefy  gelatin  rapidly  or  in  the 
Actinomycetes.  Conn  tries  to  account  for  the  noted  phenomenon 
by  assuming  two  groups  of  bacteria — winter  and  summer  bacteria. 
The  latter,  he  thinks,  prevent  the  former  from  multiplying  rapidly 
in  warm  weather.  Hence,  the  increase  in  the  frozen  soil  is  due  to  the 
depressing  effect  of  the  cold  upon  the  summer  bacteria.  There  is, 


FACTORS  INFLUENCING  NUMBER 


163 


however,  the  possibility  that  the  freezing  breaks  up  the  clumps  of 
bacteria  and  the  noted  increase  is  apparent  and  not  real.  Little 
relationship  has  been  found  between  the  moisture  and  cropping  of  a 
soil  and  the  bacterial  count- 


Date. 

Moisture 
content. 
Per  cent. 

Number  of  colonies  per  gram  of  dry 
soil. 

Remarks. 

Non-spore-form- 
ing bacteria. 

Other  colonies  — 
spore  formers  and 
actinomycetes. 

Nov.  24,  1911     .      . 

25.0 

22,500,000 

5,500,000 

Jan.    13,  1912     .      . 

40.0            28,500,000 

7,500,000 

Frozen     8  days. 

Jan.    23,  1912     .      . 

37.0 

31,000,000 

9,000,000 

Frozen  18  days. 

Feb.    13,  1912     .      . 

43.0             11,000,000 

10,000,000 

Frozen  39  days. 

Mar.     1,  1912     .      . 

37.0             20,500,000 

6,500,000 

Frozen  57  days. 

Apr.   24,  1912     .      . 

23.4             18,000,000 

4,500,000 

May     6,  1912     .      . 

.40.0            20,000,000 

9,000,000 

June     5,  1912     .      . 

20.4             19,200,000 

4,500,000 

Sept.  23,  1912     .      . 

22.5            28,500,000 

10,500,000 

Oct.    25,  1912    .      . 

24.5 

14,500,000 

5,500,000 

Dec.     3,  1912    .      . 

26.2 

28,500,000 

6,500,000 

Jan.    15,  1913    .      . 

39.5 

14,000,000 

6,000,000 

Frozen     7  days. 

Feb.      5,  1913     .      . 

22.0 

22,300,000 

7,700,000 

Frozen     4  days. 

Feb.    14,  1913     .      . 

26.2 

44,000,000 

10,000,000        Frozen  13  days. 

Mar.  11,  1913    .      . 

38.8 

22,000,000 

7,000,000 

Partly  frozen. 

Apr.      4,  1913     .      . 

22.8             19,300,000 

7,700,000 

July    10,  1913    .      . 

17.0             16,800,000 

5,200,000 

Nov.  26,  1913    .      . 

21.5             11,800,000 

4,200,000 

Dec.   15,  1913    .      . 

19.6              7,800,000 

3,200,000 

Jan.    16,  1914    .      . 

31.2 

15,500,000 

3,500,000 

Frozen     9  days. 

Jan.    30,  1914    .      . 

24.6 

26,000,000 

7,000,000 

Thawed  1  day. 

Feb.      7,  1914     .      . 

27.7 

22,700,000 

7,300,000 

Partly  frozen. 

Feb.    26,  1914    .      . 

32.0 

31,000,000 

7,000,000 

Frozen  21  days. 

Apr.    15,  1914    .      .    ! 

20.0 

13,700,000 

7,300,000 

Apr.    29,  1914    .      . 

20.0 

11,800,000 

4,200,000 

Aug.     7,  1914     .      .    I 

9.0 

5,000,000 

4,000,000 

Aug.  19,  1914    .      .   j 

20.0 

18,000,000 

4,700,000 

The  number  of  organisms  in  the  soil  vary  greatly  with  the  depth. 
Due  to  the  lack  of  moisture  and  the  germicidal  action  of  light,  the 
number  of  bacteria  in  the  uppermost  inch  or  two  of  soil  is  lower  than 
in  the  layers  of  soil  immediately  below.  Beyond  the  depth  of  eight 
or  nine  inches,  the  number  diminish  rapidly,  and  in  humid  regions 
the  number  below  two  feet  is  extremely  low.  This  is  illustrated  by 
results  reported  by  Chester: 

Depth.  Number  of  colonies. 

2  inches 981,000 

4  inches  . 1,632,000 

6  inches 1,623,000 

12  inches 73,000 

18  inches 21,000 

24  inches 4,000 

The  conditions,  however,  are  quite  different  in  the  arid  regions 
where  we  have  the  slow  formation  of  clay  substances  in  the  soil, 
and,  therefore,  the  absence  of  the  cementing  substances  in  the  soil. 


164  THE  SOIL  FLORA 

This  results  in  greater  aeration,  and  hence  often  ideal  conditions 
for  bacterial  growth  to  a  great  depth.  Moreover,  the  roots  in  search 
for  water  penetrate  to  a  great  depth  in  the  soils  of  the  arid  regions. 
This  results  in  an  aeration  of  the  soil  and  the  supplying  of  organic 
matter  to  bacteria  at  a  great  depth.  Lipman  found  the  ammonify- 
ing organisms  at  all  depths  to  the  tenth  foot  and  at  times  the  nitrify- 
ing organisms  to  a  depth  of  eight  feet.  The  nitrogen-fixing  organ- 
isms seldom  occurred  below  the  third  or  fourth  foot.  I  have  found 
great  numbers  of  bacteria  in  both  dry-farm  and  irrigated  soils  of 
the  arid  regions  in  the  second  and  third  foot.  The  average  of  several 
hundred  such  determinations  is  given  below : 

Number  of  colonies. 
Depth.  Irrigated  soil.  Dry-farm  soil. 

Ifoot 6,240,000  4,372,000 

2  feet 1,760,000  1,267,000 

3  feet 1,147,000  1,174,000 

The  larger  number  found  in  the  irrigated  soil  is  due  to  the  pres- 
ence of  a  better  supply  of  organic  matter  and  not  to  the  moisture 
supplied. 

Kinds  of  Microorganisms  in  Soil.— The  work  so  far  done  in  this 
field  clearly  establishes  the  fact  that  soils  have  a  definite  bacterial 
flora  as  do  water  and  cheese.  The  work  done  on  the  soil  so  far  is 
meager  and  has  been  carried  on  by  a  few  investigators— Hiltner  and 
Stormer  in  Germany,  and  Chester,  Harding,  and  Conn  in  this 
country.  By  far  the  best  and  most  extensive  piece  of  work  is  that 
of  Conn,  and  it  is  on  his  work  that  the  main  points  of  the  following 
are  based. 

Hiltner  and  Stormer  found  that  normally  soil  contains  5  per  cent, 
of  liquefiers,  70  per  cent,  of  non-liquefiers,  and  20  per  cent.  Strepto- 
thrix.  The  5  per  cent,  liquefiers  include  the  B.-subtilis  and  Ps. 
fluorescens  groups.  Chester  showed  that  the  relative  abundance  of 
these  three  groups  is  nearly  constant  in  normal  soil  and  that  any 
external  influence  which  disturbed  the  equilibrium  of  the  soil  flora 
would  be  indicated  by  a  change  in  the  relative  abundance  of  these 
three  groups.  This  conception  of  the  soil  microorganisms  as  being 
normally  in  a  state  of  equilibrium  has  proved  of  considerable  value 
in  interpreting  soil  phenomena. 

Conn  divides  soil  bacteria  into  the  following  groups: 


1.  Spore-producers 

2.  Non-spore-producers 

Liquefaction  rapid 
Liquefaction  slow  or  none 
Rods 

Yellow  chromogenic 


Non-chromogenic 
Cocci 
3.  Actinomycetes  Actinomycetes 


"Rapid  liquefiers" 


"Slow  growers" 


CULTURAL  CHARACTERISTICS  165 

He  found  the  relative  number  of  these  organisms  occurring  in 
soils  to  be  as  follows: 

1.  From  5  to  10  per  cent,  spore-formers  (the  B.  subtilis  group). 
Nearly  all  the  colonies  of  these  bacteria,  however,  seem  to  come  from 
spores  instead  of  from  active  organisms. 

2.  Under   10  per   cent,   rapidly  liquefying,   non-spore-forming, 
short  rods  with  polar  flagella  (principally  Ps.  fluorescens) . 

3.  From  40  to  75  per  cent,  slowly  liquefying  or  non-liquefying, 
non-spore-forming,  short  rods. 

4.  A  few  micrococci.    In  cultural  characteristics  these  are  almost 
identical  with  the  last  mentioned  group. 

5.  From  12  to  50  per  cent.  Actinomycetes. 

The  most  abundant  spore-formers  found  in  the  soil  and  described 
by  Conn  were  B.  megatherium  De  Bary,  B.  mycoides  Fliigge,  B. 
cereus  Frankland,  and  B.  simplex  Gottheil. 

"B.  megatherium  De  Bary,  1884.— This  species  is  to  be  distin- 
guished from  B.  mycoides  and  B.  cereus  by  the  larger  average  size 
of  its  spores,  by  its  poor  growth  in  liquid  media,  its  failure  to  grow 
in  the  closed  arm  of  fermentation  tubes  of  dextrose  broth,  and  by 
its  comparatively  slow  liquefaction  of  gelatin. 

"Morphology.— Very  young  cultures  (under  twelve  hours)  consist 
of  large  rods  about  1  to  1.5  M  in  diameter  and  about  3  to  6  /* 
long.  They  often  occur  in  chains  with  connecting  threads  be- 
tween the  rods,  resembling  strings  of  sausages.  In  older  cultures 
the  rods  generally  become  swollen  and  are  sometimes  full  of  highly 
refractive  globules  (fat  drops?).  One  of  the  most  distinctive 
characteristics  is  the  presence  in  cultures  a  day  or  more  old  of 
large  ovoid  bodies  about  2  by  4 /z  in  size,  which  seem  to  have 
heavy  walls  and  stain  much  more  lightly  than  the  young  rods. 
Only  the  young  rods  are  motile,  and  they  are  not  vigorously  so. 
The  flagella  are  difficult  to  stain,  and  the  best  preparations  made 
show  comparatively  few  flagella  on  each  rod.  Spores  are  formed  in 
the  center  of  the  rods  and  immediately  become  free  from  all  trace  of 
the  sporangium  wall.  They  are  oval  to  ovoid  (or  occasionally 
reniform)  and  vary  considerably  in  size,  from  1.3  to  2  //,  in  diameter 
and  from  1.5  to  3  ju  in  length,  both  extremes  often  occurring  in  the 
same  preparation. 

"Cultural  Characteristics.— Growth  in  broth  flocculent  or  none, 
with  no  surface  growth.  Gelatin  colonies  under  10  mm.  in  diameter, 
center  white,  opaque,  flocculent  or  granular,  surrounded  by  a  clear 
liquefied  zone.  Growth  on  agar  streak  cultures  smooth,  soft,  glisten- 
ing, cream-color,  typically  with  minute  drop-like  areas  of  lighter 
color. 

"Physiology.— The  typical  group  number  is  B.  111. 44420 ?4.  As 
indicated  by  this  group  number,  there  is  ordinarily  no  growth  in 
sugar  and  glycerin  broths.  This  does  not  mean,  however,  that  no 


166  THE  SOIL  FLORA  . 

acid  is  produced  from  sugar  or  glycerin.  Some  cultures  have  pro- 
duced growth  and  have  acidified  one  or  even  all  of  the  sugars.  This 
suggests  that  the  irregularity  may  be  due  to  the  poor  growth  in 
liquids,  tested  on  "slants"  of  litmus  agar,  in  fact,  B.  megatherium 
has  been  found  to  produce  acid  from  dextrose  and  sucrose  quite 
regularly.  Similarly,  its  poor  growth  in  broth  raises  a  doubt  as  to 
whether  the  second  figure  of  its  group  number  (denoting  it  to  be  a 
strict  ae'robe)  may  be  correct;  for  it  grows  so  poorly  even  in  the  open 
arm  of  a  fermentation  tube  that  its  failure  to  grow  in  the  closed  arm 


4-  0000000 

FIG.  24.— B.  megatherium.      X  1000  diameters.     (After  Conn) 

does  not  necessarily  prove  its  inability  to  grow  in  the  absence  of 
oxygen.  It  is  also  possible  that  its  failure  to  reduce  nitrates  may 
be  due  merely  to  the  fact  that  it  grows  poorly  in  nitrate  broth. 

"B.  mycoides  Fliigge,  1886. —This  is  the  most  easily  recognized  of 
all  the  soil  bacteria.  It  can  readily  be  distinguished  by  its  rhizoid 
growth  on  agar. 

"Morphology.— Young  cultures  consist  of  rods  about  0.8  to  1.3  by 
2  to  6  IJL.  They  occur  in  long  chains  which  often  lie  parallel  and 
show  false  branching,  an  arrangement  which  gives  the  colonies 


c. 
00  00060 

FIG.  25.— B.  mycoides.      X  1000  diameters.     (After  Conn) 

their  rhizoid  structure.  The  very  young  rods  are  apparently 
slightly  motile,  but  no  success  has  been  obtained  in  staining  flagella. 
Gottheil  describes  several  peritrichic  flagella.  In  older  cultures, 
highly  refractive  globules  that  do  not  take  ordinary  stains  (probably 
fat  drops)  appear  within  the  rods,  particularly  if  growing  on  dex- 
trose agar,  sometimes  causing  the  rods  to  swell  to  extremely  large 
size  and  to  lose  all  resemblance  to  their  original  form.  The  lightly 
stained  ovoid  bodies  that  characterize  B.  megatherium  have  never 
been  observed.  Spores  are  borne  centrally  and  the  remnants  of  the 


CULTURAL  CHARACTERISTICS  167 

sporangium  wall  persist  for  some  little  time  at  either  end  of  the 
spores.  Spores  are  oval  to  cylindrical  1.0  to  1.6  by  2  to  2.5  //  often 
in  fairly  long  chains. 

"Cultural  Characteristics.— Growth  in  broth  vigorous,  flocculent, 
with  no  persistent  surface  growth.  Gelatin  colonies  rapidly  liquefy- 
ing, filamentous  to  rhizoid.  Growth  on  agar  streak  rhizoid,  mostly 
beneath  the  surface  of  the  medium. 

"Physiology.— The  typical  group  number  is  B.  121. 23230 ?2.  It 
shows  less  variation  than  does  the  group  number  of  B.  megatherium, 
probably  because  B.  mycoides  grows  better  in  the  media  used  for 
making  the  tests.  The  same  acid  reactions  are  obtained  in  broth 
culture  and  on  litmus  agar. 

"B.  cereus  Frankland,  1887. —This  type  can  be  distinguished 
from  B.  megatherium  by  the  smaller  size  of  its  spores  and  by  its 
more  vigorous  growth  in  liquid  media,  and  from  B.  mycoides  by  the 
absence  of  rhizoid  growth  on  agar. 


00 
FIG.  26.— B.  cereus.       X  1000  diameters.     (After  Conn) 

"Morphology.— In  morphology  it  is  scarcely  to  be  distinguished 
from  B.  mycoides.  Young  rods  are  0.8  to  1.3  by  2.  to  6  /*  form- 
ing long  chains,  but  unlike  B.  mycoides  they  are  very  actively 
motile  and  are  easily  shown  to  be  surrounded  with  numerous  flagella. 
Older  rods  are  often  swollen  and  contain  unstained  globules.  Oval 
to  cylindrical  spores,  1.2  to  1.6  by  2.  to  2.5  M,  are  produced  cen- 
trally, retain  the  remnants  of  the  sporangium  wall  for  a  short  time 
and  often  cling  together  in  chains. 

"Cultural  Characteristics.— Growth  in  broth  vigorous,  with  uniform 
turbidity,  sediment  and  a  surface  pellicle.  Gelatin  colonies  quite 
large,  ordinarily  round,  with  entire  margin,  and  covered  with  a 
pellicle  that  generally  shows  concentric  rings,  although  under  some 
conditions- the  colonies  are  filamentous  and  resemble  those  of  B. 
mycoides.  Growth  on  agar  streak  raised,  ordinarily  rugose,  soft 
to  membranous,  generally  dull;  never  rhizoid  or  beneath  the  surface 


168  THE  SOIL  FLORA 

of  the  medium  like  B.  mycoides  or  full  of  clear  drop-like  areas  like 
B.  megatherium. 

"Physiology.— The  typical  group  number  is  the  same  as  that  for 
B.  mycoides,  B.  121.23230?2,  although  considerable  variation  has 
been  observed,  particularly  in  the  production  of  acid  from  sugars 
and  from  glycerin.  In  the  earlier  work,  two  subtypes  were  recog- 
nized, basing  the  distinction  upon  the  production  of  acid  from 
glycerin;  but  no  such  distinction  is  now  recognized,  because  of  the 
inconsistent  results  obtained  in  regard  to  acid  production.  Dextrose 
is  always  acidified  by  this  organism;  sucrose  is  generally  acidified; 
glycerin  less  frequently,  and  lactose  very  seldom.  A  strain  that 
acidifies  lactose  has  always  been  found  to  produce  acid  from  all 
three  of  the  other  compounds.  The  production  of  acid  from  lactose 
may  be  a  better  basis  for  subdividing  the  type  than  acid-production 
from  glycerin.  Out  of  130  cultures  studied,  19  acidified  lactose; 
but  it  seems  unwise  to  consider  them  as  constituting  a  separate 
species,  in  view  of  the  variation  that  has  been  found  when  cultures 
have  been  retested. 

"Although  many  of  the  spore-formers  are  active  ammonifiers  in 
solution  and  occur  in  soils  in  comparatively  large  numbers,  yet 
it  is  doubtful  if  they  play  any  very  important  role  in  soil  fertility. 

"Although  of  considerable  importance,  except  for  the  nitrifiers  and 
some  other  organisms  concerned  with  the  transformation  of  nitrogen, 
scant  consideration  has  been  given  to  any  non-spore-forming  bac- 
teria found  in  soil." 

Ps.  fluorescens  which  belongs  to  this  group  is  described  by  Conn 
as  follows: 

"Ps.  fluorescens  (Flugge)  Migula.— The  most  striking  character- 
istic of  this  type  is  its  fluorescence,  which  is  observed  in  broth, 
beef-extract-peptone  agar,  and  sometimes  in  gelatin.  Ability  to 
produce  fluorescence  is  often  lost,  however,  and  then  the  type  must 
be  recognized  by  other  characteristics,  such  as  rapid  liquefaction 
of  gelatin,  uniform  turbidity  in  broth,  cloudy,  structureless  colony 
in  gelatin,  and  acid  production  from  dextrose. 

"Morphology.— Rods  0.4  to  0.8  by  0.8  to  1.5  microns  in  old  cultures 
nearly  the  same  shape  and  size  as  in  young  cultures.  Flagella  3  to 
6,  arranged  in  a  clump  at  one  pole.  Motility  great.  Rods  do  not 
form  chains. 

1 '  Cultural  Characteristics. — G  ood  growth  in  broth ;  no  surface  growth, 
uniform  turbidity,  causing  distinct  cloudiness  of  medium;  sediment 
scant  or  none.  Gelatin  colonies  liquefying  with  great  rapidity; 
round  to  irregular  in  shape,  cloudy,  structureless,  occasionally 
fluorescent.  Growth  on  agar  streak  cultures,  smooth,  soft,  glistening 
generally  causing  the  medium  to  show  a  green  fluorescence. 

"Physiology.— The  typical  group  number  is  Ps.  211.2332133. 
Fairly  consistent  results  can  be  obtained  in  determining  its  group 


CULTURAL  CHARACTERISTICS  169 

number,  although  certain  variations  occur.  Occasionally  there  is 
no  acid  from  dextrose.  Some  of  the  cultures  reduce  nitrates.  The 
power  of  producing  fluorescence  is  often  lost.  These  variations  may 
indicate  the  existence  of  separate  species  that  are  now  grouped 
under  this  one  head." 

Actinomyces  from  12  to  50  per  cent,  of  the  organisms  found  in 
soil  are  the  Actinomycetes.  This  genus,  the  Actinomyces  Harz,  em. 
Gasperini,  is  characterized  by  the  possession  of  a  mycelium  composed 
of  hyphse  which  show  true  branching,  like  those  of  the  higher  fungi 
(seldom  measuring  over  2  microns  in  diameter),  and  judging  by 
their  staining  reactions  resemble  true  bacteria  in  their  protoplasmic 
properties.  Their  growth  is  not  wholly  within  the  agar  or  gelatin 
medium  upon  which  they  have  been  inoculated;  for  when  condi- 
tions favor,  an  aerial  mycelium  is  produced.  In  the  aerial  mycelium 
"conidia"  are  formed.  These  conidia  are  sometimes  round,  some- 
times oval,  and  sometimes  rod-shaped.  They  resemble  bacteria 
closely  in  size,  shape,  and  staining  properties.  They  are  generally 
between  0.6  and  1.5  microns  in  diameter,  and  if  oval  or  rod-shaped, 
between  1  and  2  microns  long.  They  stain  readily  with  ordinary 
bacterial  stains,  and  in  a  microscopic  preparation  which  does  not 
contain  any  hyphse,  often  cannot  be  distinguished  from  true  bac- 
teria. In  many  cases  deep-stained  granules  show  at  the  poles, 
strongly  suggestive  of  the  metachromatic  granules  of  the  diphtheria 
organism.  According  to  Sanfelice,  some  of  the  actinomycetes  are 
acid-fast  like  the  tubercle  organism.  The  diphtheria  and  tubercle 
organisms,  moreover,  sometimes  produce  branching  forms,  and  some 
writers  place  these  two  organisms  in  the  same  group  with  Actino- 
myces. 

The  growth  of  actinomycetes  on  solid  media  is  very  characteristic. 
The  mass  of  growth  is  generally  of  a  tough,  leathery  consistency, 
sometimes  smooth,  sometimes  wrinkled,  and  often  piled  high  above 
the  surface  of  the  medium.  Often  the  mass  is  brilliantly  col- 
ored, and  the  color  produced  varies  greatly  with  differences  in  the 
composition  of  the  medium,  but  with  constant  composition  of  the 
medium,  the  color  of  the  growth  may  be  characteristic  of  the  species. 
The  aerial  mycelium,  often  produced  above  this  growth,  may  also 
be  brilliantly  colored  and  of  an  entirely  different  color  from  the  mass 
of  growth  beneath  it.  Sometimes  the  aerial  hyphse  are  short  and 
give  the  growth  a  chalky  or  mildewy  appearance;  but  often  they  are 
long  enough  to  cover  the  growth  with  a  light,  delicate  nap,  1  or  2  mm. 
thick.  Some  species  produce  pigments  that  diffuse  through  the 
medium,  coloring  it  gray,  yellow,  brown,  red,  blue,  or  green.  The 
color  varies  with  the  species  and  with  the  composition  of  the  medium. 
It  is  not  so  definitely  characteristic  of  the  species  as  is  the  color  of 
the  growth  itself  or  of  the  aerial  mycelium;  but  with  a  medium  of 
constant  composition,  the  color  produced  is  of  considerable  value 


170  THE  SOIL  FLORA 

in  the  recognition  of  species.  On  gelatin  there  is  less  diversity  of 
growth  than  on  agar.  The  growth  is  generally  gray,  brown,  or 
colorless;  the  aerial  mycelium  is  often  lacking,  and  if  present  is 
white,  gray,  or  colorless;  and  if  the  medium  itself  is  colored,  it 
generally  becomes  a  reddish  brown. 

The  growth  in  liquid  media  is  also  characteristic.  The  medium 
remains  clear  except  for  small  colonies  that  may  sink  to  the  bottom, 
remain  in  suspension,  float  on  the  surface,  or  adhere  to  the  walls 
of  the  tube.  The  surface  colonies  often  grow  together  and  become 
covered  with  a  mass  of  aerial  mycelium,  sometimes  forming  a  firm, 
wrinkled  membrane  that  strongly  suggests  the  surface  membrane 
of  the  tubercle  organism  growing'  on  broth.  Pigments  are  often 
produced  in  liquid  culture,  the  pigment  varying  with  the  composition 
of  the  medium  and  with  the  species  growing  in  it. 

Nearly  all  liquefy  gelatin  and  ammonify  proteid,  Miinter  main- 
taning  that  ammonification  is  their  chief  function.  Nitrate  reduc- 
tion has  often  been  observed,  as  has  the  decomposition  of  cellulose. 
Some  are  animal  pathogens,  and  at  least  one  a  plant  pathogen. 
Other  important  physiological  activities  will  undoubtedly  be  worked 
out  when  the  technic  for  studying  them  is  further  developed.  It 
is  not  impossible  that  they  are  as  diverse  in  physiology  as  are  the 
true  bacteria. 

One  of  the  most  common  characteristics  of  many  members  of  this 
group  is  their  peculiar  odor.  They  have  a  pungent,  musty  odor, 
difficult  to  describe,  but  impossible  to  mistake  after  once  having  it 
brought  to  the  attention.  It  is  sometimes  spoken  of  as  an  earthy 
odor,  but  it  would  be  more  correct  to  say  that  soil  often  has  an 
actinomyces-odor,  as  the  odor  of  the  cultures  is  much  stronger  than 
that  of  soil,  and  the  soil  odor  is  undoubtedly  due  to  the  actinomy- 
cetes  it  contains.  The  odor  seems  to  be  associated  with  the  aerial 
conidia,  and  does  not  seem  to  be  produced  by  cultures  that  do  not 
possess  aerial  mycelium.  Not  all  species  of  Actinomyces  have  this 
odor,  however,  even  when  an  abundant  aerial  mycelium  is  produced. 

Various  functions  have  been  ascribed  to  the  actinomycetes— 
ammonification,  nitrate  reduction,  and  cellulose-decomposition— but 
enough  work  has  not  been  done  to  enable  a  definite  statement  as 
to  whether  these  are  their  functions  in  the  soil.  Conn  has  demon- 
strated that  the  addition  of  grass  roots  to  a  soil  materially  increases 
this  group  of  organisms  and  Waksman  and  coworkers  consider 
that  inasmuch  as  the  actinomyces  are  strong  cellulose  decomposers 
and  weak  producers  of  ammonia  their  probable  role  in  soil  fertility 
lies  in  the  formation  of  humus. 

REFERENCES. 

Conn,  H.  Joel:  Soil  Flora  Studies,  New  York  Agr.  Exp.  Sta.  Tech.  Buls.,  57,  58,  59, 
and  60. 

Waksman,  Silman  A.:  Cultural  Studies  of  Species  of  Actinomyces.  Soil  Science, 
1919,  viii,  71-215. 


CHAPTER  XVII. 

MINERALIZATION  AND  SOLVENT  ACTION  OF 
BACTERIA. 

SOILS  are  the  earthy  material  in  which  plants  have  their  anchorage, 
and  from  which  they  obtain  their  water  and  part  of  their  food. 
They  are  in  reality  disintegrated  rock  intimately  mixed  throughout 
with  varying  quantities  of  decaying  plant  and  animal  residues. 
They  are  derived  from  the  native  rocks  by  a  complex  process  known 
as  weathering.  The  agents  at  work  to  bring  this  about  are  changes 
of  temperature,  the  action  of  air,  water,  ice,  and  plant  and  animal 
life. 

Bacteria  as  Soil  Formers.— Early  in  the  history  of  soil  formation 
bacteria  appear  and  play  an  essential  part  in  rendering  the  soil 
fertile.  Their  life  activities  result  in  the  production  of  carbon 
dioxid,  organic  and  inorganic  acids,  and  alkalies.  These  in  turn 
react  with  the  constituents  of  the  rock  particles,  thereby  changing 
their  solubility.  When  water  becomes  charged  with  substances 
from  the  soil,  its  solvent  powers  are  greatly  increased.  Especially 
is  this  true  when  it  becomes  filled  with  carbon  dioxid  either  from  the 
atmosphere  or  from  the  decay  of  plants  and  animals. 

Common  limestone  is  one  of  the  rocks  most  actively  attacked  by 
carbonated  water;  none  are  wholly  resistant  to  its  action.  Even 
quartz  is  slowly  dissolved.  Granite  and  related  rock  are  rather 
quickly  acted  on  by  water  due  to  the  feldspar  minerals  which  it 
contains.  The  bases— potash,  soda,  lime,  and  alumina— are 
dissolved  out.  The  last  is  deposited  as  clay,  the  first  as  beneficial 
or  injurious  soil  constituents,  depending  on  the  kind  and  the  con- 
centration left  in  a  particular  soil.  In  a  similar  manner  the  sulphur, 
iron,  and  phosphorus  of  the  soil  are  changed  to  available  forms. 

Moreover,  bacteria  play  a  very  important  part  in  the  mineraliza- 
tion of  plant  and  animal  residues  which  continually  find  their  way 
into  the  soil.  The  phosphorus,  sulphur,  iron,  calcium,  magnesium, 
and  potassium  in  the  plants  and  animals  are  mainly  in  the  form  of 
organic  compounds  and  as  such  are  not  available  to  other  plants. 
Bacteria  act  upon  them,  liberating  carbon  dioxid,  ammonia,  hydrogen 
sulphid  or  sulphur  with  the  liberation  of  the  plant-food.  In  this 
manner,  the  biological  activities  become  of  the  utmost  importance  in 
the  transformation  and  migration  of  mineral  substances  in  Nature. 
The  bacteria  act  merely  as  the  link  between  the  living  and  the  dead. 


172  MINERALIZATION  AND  SOLVENT  BACTERIA 

Calcium  and  magnesium  occur  in  soils  mainly  as  carbonate,  sul- 
phate, silicate,  and  as  the  cation  in  the  salts  of  organic  acids  which 
have  resulted  during  the  breaking  down  of  the  organic  plant  and 
animal  residues. 

Soil  bacteria  in  their  life  processes  are  continually  forming  large 
quantities  of  carbon  dioxid,  nitrous,  nitric,  and  sulphuric  acid, 
together  with  organic  acids  which  are  in  the  main  combined  with 
calcium  or  magnesium  of  the  soil,  with  the  formation,  often,  of  a 
soluble  compound.  The  waters  carry  these  to  the  lakes,  seas,  and 
oceans— there  to  be  taken  up  by  marine  life.  In  the  course  of  time 
these  are  deposited  as  coral  reefs,  chalk  cliffs,  and  marl  beds.  At 
times  the  speed  with  which  the  lime  is  taken  from  the  waters  by 
marine  life  is  faster  than  it  is  carried  into  a  lake  by  its  tributaries. 
The  result  is  that,  in  spite  of  the  evaporation  and  concentration 
which  is  going  on,  the  main  body  of  water  contains  less  lime  than 
does  its  tributaries.  This  is  the  case  with  Bear  Lake,  Utah,  the 
tributaries  of  which  have  an  average  lime  content  of  101.7  parts 
per  million,  whereas  the  lake  contains  only  13.2  parts  per  million. 

Calcium  Carbonate.— The  loss  of  calcium  carbonate  from  a  soil 
varies  with  (1)  the  methods  of  agriculture,  intense  methods  increas- 
ing the  loss;  (2)  with  the  application  of  animal  manures  and  green 
manures,  which  increases  the  bacterial  activity  and  also  the  solubility 
of  the  calcium  carbonate;  (3)  with  the  addition  of  commercial  fertil- 
izers added  to  a  soil  which  hasten  the  loss  of  calcium  in  drainage 
water. 

The  carbon  dioxid  generated  by  bacteria  reacts  with  the  calcium 
carbonate  forming  the  much  more  soluble  calcium  bicarbonate: 
or 

CaCO3     +     COa  '  +     H2O      =     Ca(HCO3)2 

Ammonium  sulphate  resulting  either  from  ammonification  or  from 
the  addition  of  a  fertilizer  changes  the  calcium  from  an  insoluble 
to  a  soluble  form : 

(NH4)2SOi  +  2CaCO3  +  4O2   =  Ca(NO3)2  +  CaSO4  +  4H2O   +  2CO2 
(NH4)2SO4     +     CaCO3     =     (NH4)2CO3     +     CaSO4 

The  addition  of  acid  phosphate  or  potassium  chlorid  also  helps 
deplete  the  soil  of  its  calcium  carbonate: 

CaH4(PO4)2     +     2CaCO3     =     Ca3(PO4)2     +     2H2O     +     2CO2 
2KC1      +     CaCOs       =     K2CO3  +     CaCl2 

The  absolute  amount  of  calcium  and  magnesium  lost  from  a  soil 
varies  with  the  aridity  of  a  region  as  well  as  with  the  composition 
of  the  soil.  Hall  estimates  that  the  annual  loss  from  the  Rotham- 
sted  soil,  which  contains  about  3  per  cent,  of  calcium  carbonate,  is 
from  800  to  1000  pounds  an  acre  annually,  whereas  in  some  parts 


PHOSPHORUS  173 

of  Scotland,  where  liming  has  been  practised  for  some  time,  the  loss 
is  from  500  to  600  pounds.  In  this  country,  where  liming  is  necessary, 
the  farmers  usually  provide  for  a  loss  of  400  pounds  an  acre  annually. 
Bacteria  are  also  responsible  for  the  restoration  of  varying 
amounts  of  carbonates.'  In  the  weathering  of  complex  silicates, 
carbonates  and  silicic  acid  may  be  formed  in  considerable  quantities : 

CaAl2Si2O8     +     CO2     +     2H2O      =     Al2Si2O5(OH)4     +     CaCOs 

According  to  Nadson,  soil  bacteria  may  cause  the  formation  of 
calcium  carbonate  from  calcium  sulphate  through  the  reacting  of 
ammonium  carbonate  formed  in  the  decay  of  protein  substances 
with  calcium  sulphate: 

(NH4)2CO3     +     CaSO4     =     (NH4)2SO4     +     CaCO3 

Or  even  after  the  sulphate  has  lost  its  oxygen  through  the  action  of 
reducing  bacteria,  calcium  carbonate  may  be  formed  through  the 
action  of  carbon  dioxid  and  water  on  the  calcium  sulphid : 

CaS     +     CO2     +     H2O      =     CaCO3     +.    H2S 

Denitrifying  bacteria  may  act  on  calcium  nitrate  with  the  forma- 
tion of  calcium  carbonate: 

2Ca(NO3)2     +     2CO2    -»     2CaCO3     +     2N2     +     5O2 

Calcium  carbonate  may  also  be  formed  in  the  soil  due  to  the 
action  of  bacteria  upon  humates  and  calcium  salts  of  simpler  organic 
acids : 

(RCOO)2Ca     =     CaCOs     +     RCOR 

Cunningham  has  demonstrated  that  Azotobacter  chroococcum  is 
capable  of  growing  in  solution  of  calcium  oxalate  with  the  formation 
of  calcium  carbonate,  as  were  also  six  other  types  of  organisms 
isolated  by  him.  The  presence  of  oxygen  is  essential  for  the  process. 
He  considers  that  an  equilibrium  is  set  up  by  which  the  withdrawal 
of  calcium  carbonate  is  balanced  by  the  results  of  another  set  of 
reactions  which  restores  the  base  to  the  soil.  This  enables  many 
soils  which  contain  only  very  small  quantities  of  lime  to  retain  their 
neutral  reaction  and  so  to  produce  fair  crops.  This,  however,  is  not 
always  the  case,  as  is  witnessed  by  the  acid  soils  occurring  in  many 
agricultural  districts. 

Phosphorus.— Phosphorus  occurs  mainly  in  the  form  of  the  calcium, 
iron,  or  aluminum  phosphate;  in  any  soil  the  quantity  soluble  is 
small.  Moreover,  as  soluble  phosphorus  compounds  are  applied  to 
the  soil  they  become  fixed  as  insoluble  compounds.  Hence,  the  loss 
through  leaching  of  this  element  from  the  soil  is  small  under  any 
conditions. 


174  MINERALIZATION  AND  SOLVENT  BACTERIA 

There  are  also  varying  amounts  of  organic  phosphorus  in  soil. 
This  occurs  in  the  form  of  lecithin,  phospho-proteins,  and  nucleo- 
proteins.  Little  has  been  done  to  determine  the  action  of  bacteria 
upon  these  compounds,  but  it  is  to  be  expected  that  they  would  be 
hydrolyzed  by  bacteria  as  they  are  by  ferments. 

Lecithin  yields  on  hydrolysis  glycerin,  two  molecules  of  fatty 
acid,  phosphoric  acid,  and  cholin: 


4H2O      =     Ci8H34O2     +     C16H32O2     +     C3H8O3     + 
lecithin  water  oleic  acid        palmitic  acid        glycerol 

H3PO4     +     C6Hi5NO2 
phosphoric  acid        cholin 

The  phospho-proteins  yield  on  hydrolysis  amino-acids  and  phos- 
phoric acid,  whereas  hydrolytic  cleavage  produces  from  nucleo- 
proteins  carbohydrates,  phosphoric  acid,  purin  and  pyrimidin  bases, 
with  the  intermediate  formation  of  nucleins  and  nucleic  acid,  as 
may  be  represented  by  the  following  scheme: 

nucleo-proteins 


proteins 

nucleins 

i 

proteins 

nucleic  acid 

carbohydrates 
pentoses 
hexoses 
unidentified 

phosphoric                   purin  bases                   pyrimidin  bases 
acid                         adenin                           thymin 
guanin                           cytosin 
xanthin                         uracil 
hypoxanthin 

Schettenhelm  has  shown  that  nearly  all  of  the  nuclein  substances 
of  feces  disappear  as  they  undergo  autoputrefaction.  He  and 
Schroeter  showed  that  bacteria  may  bring  about  a  deep  cleavage  of 
yeast  nucleic  acid.  Plenge  showed  that  some  bacteria  have  the 
power  to  liquefy  the  sodium  salt  of  nucleic  acid  from  thymus. 

It  seems  reasonable,  therefore,  to  believe  that  phosphorus  would 
be  liberated  by  soil  bacteria  in  a  somewhat  similar  manner.  It  is 
known  that  the  bacterial  flora  of  the  soil  play  a  highly  important 
role  in  rendering  the  phosphorus  of  the  inorganic  phosphates  avail- 
able to  the  higher  plant. 

Brown  found  that  twelve  out  of  twenty-three  bacteria  isolated 
from  soil  exerted  a  definite  solvent  action  on  difficultly  soluble 
plant-food.  One  organism  which  produced  no  gas  but  a  large 
amount  of  acid  showed  the  greatest  solvent  action  upon  calcium 
carbonate,  whereas  other  organisms  which  produced  gas— largely 
carbon  dioxid— but  not  as  much  acid  as  the  former,  gave  an  action 
more  marked  than  that  of  the  stronger  acid-producer  upon  the 
dicalcium  and  tricalcium  phosphates.  B.  subtilis,  B.  mycoides, 
B.  proteins  vulgaris,  and  B.  coli  communis,  as  well  as  several  agar 
cultures  from  garden  soil,  were  found  to  be  capable  of  dissolving 


PHOSPHORUS  175 

the  phosphates  of  bone  and  to  a  less  extent  that  of  mineral  phos- 
phates. The  greatest  solvent  action  was  exerted  in  media  contain- 
ing sodium  chlorid,  potassium  sulphate,  and  ferrous  sulphate.  Even 
yeast  may  be  important  in  dissolving  phosphates.  As  suggested 
by  Krober  the  life  activity  of  the  bacteria,  that  is,  assimilation  of 
phosphorus  by  the  living  organism,  probably  plays  little  or  no  direct 
part  in  dissolving  the  phosphates,  but  it  is  due  to  the  action  of  the 
organic  acids  and  of  the  carbon  dioxid  produced. 

The  acids  produced  by  bacteria  act  upon  all  phosphates,  convert- 
ing them  into  the  soluble  monophosphate,  but  the  rate  of  solution 
varies  widely  with  the  different  phosphates.  Tricalcium  phosphate 
in  precipitated  form,  dicalcium  phosphate,  and  tetracalcium  phos- 
phate of  Thomas  slag  are  much  more  rapidly  dissolved  than  the 
crystalline  or  the  so-called  amorphous  phosphates.  The  general 
reaction  is  as  follows : 

2  R  COOH     +     Ca3(PO4)2-»Ca2H2(PO4)2     +     (R  COO)2Ca 

The  reaction  takes  place  most  rapidly  in  soils  containing  large 
quantities  of  organic  matter  due  to  the  active  fermentation  taking 
place  in  such  soils. 

Grazia  considers  enzyme  action  to  play  a  part  in  the  dissolving 
of  phosphates  in  soil,  for  he  found  the  addition  of  chloroform  to  a 
soil  reduced  bacterial  activity  and  decreased  the  acid  produced,  but 
at  the  same  time  the  solution  of  phosphates  was  increased.  This  is 
in  keeping  with  the  finding  of  Bychiklin. 

The  presence  of  ammonium  chloride  and  sulphate  in  the  cultural 
media  is  especially  effective  in  increasing  the  solvent  action  of 
bacteria,  according  to  Perotti,  who  considers  the  successive  steps 
in  the  solution  or  decomposition  of  phosphorus  compounds  by 
bacteria  as  follows:  (1)  generation  of  acids,  (2)  secondary  reactions 
in  the  solution,  and  (3)  production  of  a  soluble  phosphorus  contain- 
ing organic  substance.  The  first  two  of  these  are  the  result  of 
the  activity  of  the  bacteria  on  the  phosphorus,  and  the  last  is  due 
to  the  metabolic  assimilation  of  the  microorganisms. 

The  oxidation  of  sulphur  by  soil  bacteria  may  at  times  generate 
sufficient  acid  to  play  a  very  important  role  in  dissolving  soil  phos- 
phorus. Hopkins  and  Whiting,  however,  consider  that  the  nitrite 
bacteria  are  of  the  first  importance  in  rendering  phosphorus  and 
calcium  soluble  when  they  oxidize  ammonia  into  nitrites: 

(NH4)2CO3     +     3O2     =     2HNO2     +     H2CO3     +     2H2O 

The  resulting  nitrous  acid  then  reacts  with  the  raw  rock  phosphate, 
rendering  it  soluble,  thus : 

Ca3(PO4)2     +     4HNO2     =     CaH4(PO4)2     +     2Ca(NO2)2 


176  MINERALIZATION  AND  SOLVENT  BACTERIA 

The  actual  ratio  found  showed  that  about  one  pound  of  phos- 
phorus and  about  two  pounds  of  calcium  are  made  soluble  for  each 
pound  of  nitrogen  oxidized,  aside  from  the  action  of  the  acid  radicals 
associated  with  the  ammonia.  The  carbonic  acid  would  play  an 
important  part  also  in  this  reaction: 

4H2CO3     +     Ca3(PO4)2      =     2Ca(HCO3)2     +     CaH4(PO4)2 

They  found  that  neither  ammonia-producing  bacteria  nor  nitrate 
bacteria  liberated  appreciable  quantities  of  soluble  phosphorus  from 
insoluble  phosphates. 

Whereas  this  would  readily  occur  in  soil  poor  in  calcium  carbonate, 
in  those  rich  in  calcium  carbonate  there  would  be  only  small  quanti- 
ties of  phosphorus  liberated,  according  to  Kelley.  But  where  the 
soluble  phosphorus  is  being  rapidly  removed  by  the  growing  plant, 
or  even  by  bacteria,  there  is  little  doubt  that  the  various  soil  organ- 
isms play  an  important  part  in  rendering  phosphorus  soluble,  for 
results  obtained  at  the  Utah  Experiment  Station  show  there  to  be  a 
relationship  between  the  increased  nitrification  produced  by  various 
salts,  and  the  quantity  of  water-soluble  and  organic  phosphorus  in 
the  soil.  This  is  illustrated  by  the  following  results  which  give  the 
nitric  nitrogen,  water-soluble  and  organic  phosphorus  in  a  soil  after 
various  treatmejjts,  the  untreated  soil  being  considered  as  100  per 
cent. 


PER   CENT.   NITRIC  NITROGEN  WATER-SOLUBLE   AND   ORGANIC   PHOS- 
PHORUS  OCCURRING   IN  SOIL  RECEIVING  VARIOUS   SALTS. 

Water- 
Nitric                        soluble  Organic 
nitrogen.                 phosphorus.  phosphorus. 
Treatment.                           Per  cent.                 Per  cent.  Per  cent. 

None 100.0  100.0  100.0 

312  x  10-7  mol.  MgSO4     .      .      .  101.2  105.2  111.6 

26  x  10-7  mol.  Fe2(SO4)3       .      .  102.0  94.3  142.3 

625  x  10-7  mol.  Ca(NO3)2      ..  102.0  114.3  97.5 

156  x  10-7  mot.  KNO3      .      .      .  106.4  108.1  103.3 

625  x  10-7  mol.  KC1    ....  106.5  105.8  107.3 

312  x  10-7  mol.  Mg(NO3)2     .      .  106.5  115.5  95.1 

125  x  10-6  mol.  MnCOs  .      .      .  108.4  107.5  162.6 

156  x  10-7  mol.  MnCl2     .      .      .  112.9  100.2  98.4 

78x  10-7  mol.  MnSO4    .      .      -  113.2  94.3  107.9 

13  x  10-4  mol.  FeCOg     .      .      .  117.4  105.6  94.8 

25  x  10-6  mol.  MgCl2      .      .      .  123.2  109.7  96.5 

625  x  10-7  mol.  Mn(NO3)2     .      .  125.4  84.8  87.9 

84  x  10-fi  mol.  FeCls       .      .      .  128.3  105.6  94.8 

25  x  10-6  mol.  MgCOs    -      -      -  140.7  98.2  72.2 

-      1  x  10-8  mol.  NaCl        ...  142.0  109.3  138.7 

1  x  10-8  mol.  CaCl2       .      -      -  167.2  114.9  88.2 

2  x  10-s  mol.  CaSO4     .      .      .  196.7  73.5  103.3 

Moreover,  it  is  evident  that  Azotobacter  in  their  metabolism  trans- 
form soluble  inorganic  soil  constituents  either  into  soluble  or  into 
insoluble  organic  forms.  This  is  especially  true  of  phosphorus  which 


PHOSPHORUS  177 

is  found  in  the  ash  of  these  organisms  in  such  large  quantities.  The 
phosphorus,  on  the  death  of  the  organism,  would  be  returned  to  the 
soil  in  a  readily  available  form,  for  Stoklasa  has  found  that  50  per 
cent,  of  the  nitrogen  of  these  organisms  is  nitrified  within  six  weeks, 
and  there  is  no  reason  for 'believing  that  the  phosphorus  would  be 
liberated  much  more  slowly.  Then  there  is  the  possibility  that  many 
of  the  constituents  of  the  bacterial  cell  may  become  available, 
through  the  action  of  autolytic  enzymes  without  the  intervention 
of  other  bacteria. 

It  is  further  evident  that  an  organism  which  possesses  the  power, 
when  growing  under  appropriate  conditions,  of  generating  1.3  times 
its  own  body  weight  in  carbon  dioxid  during  twenty-four  hours,  as 
does  the  Azotobacter,  must  greatly  change  the  composition  of  the 
media  in  which  it  is  growing.  Water  charged  with  carbon  dioxid 
is  a  universal  solvent  and  will  attack  even  ordinary  quartz  rock. 
Granite  and  rocks  related  to  it  are  rather  quickly  attacked,  with  the 
liberation  of  potassium  and  other  elements.  Carbonated  water 
would  act  upon  the  tricalcium  phosphate  of  the  soil  with  the  forma- 
tion of  more  readily  soluble  phosphates,  for  this  substance  is  four 
times  as  soluble  in  water  charged  with  carbon  dioxid  as  it  is  in  pure 
water: 

Ca3(PO4)2     +     2CO2     +     2H2O      =     Ca2H2(PO4)2     +     Ca(HCO3)2 

Moreover,  the  nitrogen-fixing  organisms  form,  among  other 
products,  formic,  acetic,  lactic,  butyric,  and  other  acids.  The  kind 
and  quantity  of  each  depends  upon  the  specific  organisms  and  upon 
the  substance  on  which  they  are  acting.  These  substances  are  sure 
to  come  in  contact  with  some  insoluble  plant-food  which  may  be 
rendered  soluble,  for  they  have  a  highly  solvent  action  on  the 
insoluble  phosphates.  The  resulting  salts  of  calcium  would  be 
further  attacked  by  bacteria,  with  the  formation  of  calcium  car- 
bonate. 

Whether  these  processes  will  give  rise  to  an  increase  in  the  water- 
soluble  plant-food  of  the  soil  depends  upon  whether  the  products 
of  the  second,  the  analytic  reactions,  exceed  the  products  of  the 
first,  the  synthetic  reactions.  It  must  not  be  forgotten  that, 
although  many  of  the  organic  phosphorus  constituents  may  not  be 
soluble  in  pure  water,  they  may  be  more  available  to  the  living  plant 
than  are  the  constituents  from  which  they  were  at  first  derived 
through  bacterial  activity. 

This  being  the  case,  variations  in  the  results  reported  from 
laboratory  tests  are  to  be  expected.  Stoklasa  found  that  bacterial 
activity  rendered  the  phosphorus  ot  the  soil  more  soluble,  whereas 
Severin,  in  his  early  work,  found  the  opposite  to  be  true.  Others 
have  found  that  the  solvent  action  of  bacteria  for  insoluble  phos- 
phates is  in  direct  proportion  to  the  acid  secreted  by  the  organism. 
12 


178  MINERALIZATION  AND  SOLVENT  BACTERIA 

In  a  later  work,  Severin  obtained  different  results.  He  used  three 
soils — one  sterile,  a  second  sterilized  and  inoculated  with  pure 
cultures  of  Azotobacter •,  and  a  third  sterilized  and  inoculated  with 
cultures  of  Ps.  radicicola  and  Azotobacter.  The  solubility  of  the 
phosphorus  increased  8  to  14  per  cent,  over  that  in  the  sterile  soil. 
The  acid-producing  organisms,  due  to  the  acid  secreted  and  their 
intimate  contact  with  the  soil  particles,  possess  the  power  of  dissolv- 
ing silicates.  Moreover,  arsenic  greatly  stimulates  nitrogen  fixa- 
tion, and  there  is  a  relationship  between  this  increased  bacterial 
activity  and  the  form  and  quantity  of  phosphorus  found  in  a  soil. 

Although  the  metabolic  activity  of  Azotobacter  gives  rise  to  large 
quantities  of  phosphate  solvents,  yet  these  organisms  transform 
phosphorus  into  organic  phosphorus  compounds  less  rapidly  than 
do  the  ammonifiers.  There  are,  however,  cases  in  which  bacterial 
activity  has  decreased  the  water-soluble  phosphorus  of  the  soil  and 
of  raw  rock  phosphate.  This  does  not  mean,  however,  that  it  is 
less  available,  for,  as  pointed  out  by  Truog,  the  mixing  of  floats  with 
manure  caused  an  immediate  decrease  in  the  solubility  of  the  phos- 
phorus in  0.2  per  cent,  citric  acid  solution,  yet  when  thoroughly 
mixed  with  the  feeding  area  of  the  soil  its  availability  was  increased 
to  such  an  extent  that  some  species  of  plants  were  apparently  able 
to  secure  almost  an  adequate  supply  of  phosphorus  from  this 
material.  The  addition  of  manure  to  a  soil  greatly  increased  the 
carbon-dioxid  production,  and  for  a  short  time  measurably  increased 
the  solvent  action  on  floats.  Where  there  is  for  a  time  a  decrease 
of  water-soluble  phosphorus  in  fermenting  media,  it  is  probably  due 
to  the  formation  of  phospho-proteins  within  the  bodies  of  the 
bacteria  which  would  later  be  rendered  soluble  due  either  to  further 
bacterial  activity  or  to  autolytic  enzymes. 

Sulphur.— Sulphur  is  an  essential  element  for  all  plants,  but  the 
quantity  required  is  relatively  small  and  most  soils  contain  sufficient 
for  maximum  crop  production.  It  occurs  within  the  soil  mainly  as 
sulphate  or  organic  sulphur,  and  these  substances  are  often  materially 
changed  by  bacterial  activity. 

Bacteria  act  on  sulphur  compounds  in  three  ways:  (1)  on  complex 
organic  compounds  with  the  production  of  hydrogen  sulphid  or 
mercaptans,  (2)  the  oxidation  of  sulphur  compounds  occurring  in  the 
soil,  and  (3)  the  oxidation  of  sulphur  compounds,  especially  hydrogen 
sulphid  by  the  true  sulphur  bacteria,  with  the  production  of  metallic 
sulphur,  sulphuric  acid,  and  eventually  mineral  sulphates. 

Hydrogen  sulphid  is  produced  by  the  majority  of  the  common 
laboratory  forms  of  bacteria.  Lafar  states  that  this  faculty  is  even 
very  common  among  the  pathogenic  bacteria  and  was  absent  in  not  a 
single  one  of  37  species  examined.  Other  bacteria  possess  the 
power  of  reducing  sulphates.  Beijerinck  found  in  soil  an  organism 
which  he  named  Spirillum  desutphuricans  and  which  Van  Delden 


SULPHUR  179 

later  classified  as  Microspira  desulphuricans  which  possessed  the 
power  of  reducing  sulphates.  Another  sulphate-reducing  organism 
is  Msp  aestuarii.  These  organisms  act  only  in  the  presence  of 
organic  matter: 

MSO4     +''  2C      =     2CO2     +     MS 

The  true  sulphur  bacteria  possess  a  directly  opposite  physiological 
action  to  the  reducing  bacteria.  There  are  two  genera  of  the 
true  sulphur  bacteria  recognized—  Beggiatoa  and  Thiothrix.  Beggia- 
toa  is  filamentous,  motile,  and  morphologically  resembles  the  blue- 
green  alga,  Oscillaria.  Thiothrix  is  not  filamentous  nor  motile  and 
possesses  a  sheath  and  forms  spores.  The  sulphur  bacteria  contain 
in  their  protoplasm  highly  refractive  inclusions  of  amorphous  sulphur. 
According  to  Winogradsky,  a  single  Beggiatoa  thread  used  in  a  day 
two  to  four  times  their  own  weight  of  hydrogen  sulphid  with  the 
production  of  sulphur: 

4H2S     +     2O2      =     4H2O     +     4S 

The  sulphur  seen  within  the  cell  protoplasm  is  to  be  looked  upon  as 
an  intermediate  state  in  the  oxidation  process,  for  if  the  organisms  be 
transferred  to  fresh  water  these  soon  disappear  with  the  formation 
of  sulphuric  acid: 

2S      +     3O2     +     2H2O      =     2H2SO4 

This  reacts  with  a  base,  usually  calcium  carbonate,  with  the  forma- 
tion of  calcium  sulphate : 

CaCO3     +     H2SO4     =     CaSO4     +     H2O     +     CO2 

There  are  also  organisms  in  soil  that  can  oxidize  sulphur  to  sulphuric 
acid  which  in  turn  would  act  as  a  solvent  for  plant-food.  Moreover, 
small  quantities  of  sulphur  added  to  a  soil  will  increase  ammonifica- 
tion.  It  is  likely  that  much  of  the  benefit  resulting  from  sulphur 
fertilization  is  due  to  these  factors. 

Brown  has  recently  shown  the  power  of  oxidizing  sulphur  to  vary 
with  different  soils.  Aeration  and  optimum  moisture  favor  it, 
whereas  the  addition  of  carbohydrates,  depresses  the  process.  He  has 
elaborated  a  method  of  measuring  the  speed  of  sulphur  oxidation  in 
soils  and  given  to  it  the  name  of  sulphofication. 

According  to  Lafar,  the  importance  of  the  sulphur  bacteria  in  the 
economy  of  nature  is  unmistakable.  In  cooperation  with  the  sulphate- 
reducing  bacteria  they  insure  that  the  sulphur  cycle  pursues  an  un- 
interrupted course,  the  elements  being  taken  up  by  the  higher  plants 
in  the  condition  of  sulphates  and  deposited  in  the  cells  in  the  form  of 
organic  compounds  from  which,  in  the  course  of  putrefaction,  sulphur 
is  liberated  as  hydrogen  sulphid,  and  finally  reconverted  into  sulphates 
by  the  sulphur  bacteria.  It  then  recommences  its  course  through  the 
higher  plants. 


180  MINERALIZATION  AND  SOLVENT  BACTERIA 

Iron.— The  iron  bacteria  resemble  the  sulphur  bacteria  greatly  in 
their  metabolic  activity.  The  best  known  of  these  organisms  are 
the  Crenothrix  polyspora,  Chlamydothrix  ochmcea,  and  Spirophyllum 
ferrugineum.  Winogradsky  considers  that  the  iron  is  deposited  in  the 
sheath  of  the  organisms  due  to  a  physiological  reaction,  the  organ- 
isms oxidizing  ferrous  to  ferric  compounds : 

4FeCO3     +     6H2O     +     O2      =     2Fe2(OH)6     +     4CO2 

The  energy  so  liberated  is  utilized  in  their  growth.  However,  the 
investigations  of  Molisch,  Adler,  and  Ellis  show  that  they  grow  well 
in  a  medium  devoid  of  iron  and  that  the  precipitation  of  the  iron  is 
due  to  chemical  and  mechanical  processes  independent  of  the  physio- 
logical activity  of  the  organism.  They  play  a  great  part  in  the 
deposition  of  bog-iron,  though  not  the  only  cause,  for  Molisch  con- 
siders that  well-known  physio-chemical  agencies  often  play  an 
important  part  in  the  process.  Manganese  may  at  times  be  found 
in  the  sheath  of  Crenothrix,  in  large  quantities. 

Potassium.— This  element  is  required  by  all  plants  in  compara- 
tively large  quantities,  and  the  total  supply  in  nearly  all  soils  is 
exceedingly  large  as  compared  to  crop  requirements.  Yet  potas- 
sium is  quite  extensively  used  as  a  fertilizer,  and  this  with  beneficial 
results.  This  is  due  to  the  fact  that  its  addition  to  a  soil  well 
supplied  with  available  potassium  results  in  the  liberation  of  other 
more  deficient  plant-food  elements.  Moreover,  it  may  be  applied  to 
soils  having  a  large  quantity  of  total  potassium,  but  a  small  quantity 
available  to  plants.  Therefore,  one  of  the  problems  which  is  con- 
fronting the  farmer  is  how  to  render  available  as  needed  by  plants 
the  large  supply  of  potassium  in  the  soil. 

The  potassium  occurs  in  the  soil  mainly  as  silicates  and  is  rendered 
soluble  by  the  nitrous,  nitric,  sulphuric,  acetic,  lactic,  and  butyric 
acids,  and  by  carbon  dioxid.  The  last  may  react  with  inert  potas- 
sium resulting  in  the  formation  of  available  potassium  according  to 
the  following  equation : 

A12O3K2O.     6SiO2     +     CO2     +     2H2O      =     AI2O3     2SiO2     +     2H2O     + 
K2CO3     +     4SiO2 

Hence,  the  addition  of  animal  manure,  green  manures,  com- 
mercial fertilizers,  or  even  soil  amendments  may  increase  bacterial 
activity  and  in  a  similar  degree  increase  the  soluble  soil  potassium. 

REFERENCES. 

Lafar,  Franz:     "Handbuch  der  Technischen  Mykologie,"  Dritter  Band. 
Greaves,  J.  E.,  Carter,  E.  G.:     "The  Action  of  Some  Common  Soil  Amendments" 
(Soil  Science,  vol.  vii  (1919),  pp.  121-160). 

Kossowicz,   Alex.:     "Agrikulturmykologie,    I   Bodenbakteriologie." 
Ellis,  David:     Iron  Bacteria,  London,  1919. 


CHAPTER  XVIIL 

THE  CARBON,  NITROGEN,  SULPHUR,  AND 
PHOSPHORUS  CYCLES. 

PLANTS  contain  ten  essential  elements,  and  these  elements  found 
in  the  body  of  the  plants  or  animals  today  are  the  same  as  those 
which  constituted  the  organic  world  thousands  of  years  ago.  But 
between  these  dates  they  may  have  played  many  parts,  or,  in  the 
words  of  Duncan,  "We  believe— we  must  believe  in  this  day— that 
everything  in  the  universe  of  world  and  stars  is  made  of  atoms,  in 
quantities  x,  y,  or  z,  respectively.  Men  and  women,  mice  and 
elephants,  the  red  belts  of  Jupiter  and  the  rings  of  Saturn,  are,  one 
and  all,  but  ever-shifting,  ever-varying  swarms  of  atoms.  Every 
mechanical  work  of  earth,  air,  fire,  and  water,  every  criminal  act, 
every  human  deed  of  love  or  valor;  what  is  it  all,  pray,  but  the  rela- 
tion of  one  swarm  of  atoms  to  another? 

"Here,  for  example,  is  a  swarm  of  atoms,  vibrating,  scintillant, 
martial— they  call  it  a  soldier—  and,  anon,  some  thousands  of  miles 
away  upon  the  South  African  veldt,  that  swarm  dissolves— dis- 
solves, forsooth,  because  of  another  little  swarm— they  call  it  lead. 

"What  a  phantasmagoric  dance  it  is,  this  dance  of  atoms!  And 
what  a  task  for  the  master  of  the  ceremonies!  For,  mark  you,  the 
mutabilities  of  things.  These  same  atoms  may  come  together  again, 
vibrating,  clustering,  interlocking,  combining,  and  there  results  a 
woman,  a  flower,  a  blackbird,  or  a  locust,  as  the  case  may  be.  But 
tomorrow  again  the  dance  is  ended,  and  the  atoms  are  far  away; 
some  of  them  in  the  fever  germs  that  broke  up  the  dance,  others  are 
the  green  hair  of  the  grave,  and  others  are  blown  about  the  Antipodes 
on  the  wings  of  ocean,  and  the  eternal  everchanging  dance  goes  on.'' 

In  this  building  up  and  breaking  down,  bacteria  play  an  all- 
important  part.  The  higher  plants  build  up  the  carbon  and  nitrogen 
into  complex  organic  compounds.  This  same  end  is  also  accom- 
plished to  a  lesser  degree  by  the  animals  which,  however,  mainly 
act  as  analyzers  of  organic  matter,  but  the  master  analysts  are  the 
bacteria  which  are  continually  resolving  into  simple  and  often 
elementary  constituents,  the  plant  and  animal  debris.  Were  this 
not  true,  all  the  carbon  and  combined  nitrogen  of  the  world  would 
soon  become  locked  up  in  the  dead  bodies  of  animals;  plants  would 
starve  and  die,  and  animals  would  likewise  become  extinct.  There- 
fore, bacteria  are  the  link  between  the  living  and  the  dead.  The 


182     THE  CARBON,  NITROGEN,  AND  PHOSPHORUS  CYCLES 

absence  of  bacteria  is  incompatible  with  life  on  this  earth,  or,  as 
stated  by  Pasteur,  "they  are  the  important,  almost  the  only,  agents 
of  universal  hygiene.  They  clear  away  more  quickly  than  the  dogs 
of  Constantinople  or  the  wild  beasts  of  the  desert,  the  remains  of 
all  that  has  had  life;  they  protect  the  living  against  the  dead;  they 
do  more;  if  there  are  still  living  beings,  if,  since  the  hundreds  of 
centuries  the  world  has  been  inhabited,  life  continues,  it  is  to  them 
we  owe  it." 

The  Carbon  Cycle.— Carbon  occurs  free  in  the  earth  as  coal  to  the 
extent  of  over  500  billion  tons.  Chemically  combined,  it  is  found 
in  far  larger  quantities  in  limestone,  chalk,  marble,  and  dolomite— 
rocks  which  form  such  a  considerable  portion  of  the  surface  of  the 
earth.  According  to  Pettenkofer,  a  man  weighing  154  pounds 
contains  26.4  pounds  of  carbon;  no  less  than  257  million  tons'  weight 
of  it  is,  therefore,  stored  up  in  the  bodies  of  men  and  women  living 
upon  the  earth  at  the  present  time,  to  say  nothing  of  the  far  greater 
quantities  occurring  in  the  tissues  of  trees,  plants,  and  lower  animals. 

Carbon  dioxid  occurs  in  the  atmosphere  to  the  extent  of  three 
parts  in  10,000.  This  is  the  equivalent  of  600  billion  tons  of  carbon. 
Moreover,  the  ocean  is  a  vast  reservoir  of  carbon  dioxid,  which  is 
partly  in  solution  and  partly  combined.  Between  the  surface  of 
the  sea  and  the  atmosphere  there  is  a  continual  interchange,  each  at 
times  losing  and  at  times  gaining  the  gas. 

Carbon  dioxid  is  being  added  to  the  air  from  several  sources: 
the  combustion  of  fuel,  the  respiration  of  animals,  and  the  decay  of 
organic  matter.  It  is  also  being  evolved  in  enormous  quantities 
from  mineral  springs  and  volcanoes.  Krogh  estimates  that  the 
annual  consumption  of  coal  adds  yearly  to  the  atmosphere  about 
one-thousandth  of  its  present  content  in  carbon  dioxid.  Were 
there  no  factors  offsetting  this  increase  in  atmospheric  carbon  dioxid 
animal  life  would  soon  become  extinct. 

On  the  other  hand,  there  are  two  large  factors  at  work  removing 
carbon  from  the  atmosphere — first,  the  decomposition  of  carbon 
dioxid  by  plants  with  the  liberation  of  oxygen,  and  second,  the 
consumption  of  carbon  dioxid  in  the  weathering  of  rocks.  No 
precise  valuation  can  be  given  to  either  of  these  factors,  although 
various  writers  have  attempted  to  estimate  their  magnitude.  Cook 
computes  that  leaf  action  alone  more  than  compensates  for  the 
production  of  carbon  dioxid.  Chamberlain  estimates  that  the 
amount  of  carbon  dioxid  annually  withdrawn  from  the  atmosphere 
is  1,620,000,000  tons,  and  that  the  greater  part  of  this  is  taken  up 
by  the  weathering  of  mineral.  This  is  continually  being  returned 
to  the  atmosphere  by  the  factors  considered  in  the  preceding  chapter. 
There  are  then  two  compensating  sets  of  factors— decay,  respiration, 
and  combustion  liberating  carbon;  plant  growth  and  rock  weather- 
ing fixing  it.  These  balance  each  other,  thereby  completing 


THE  NITROGEN  CYCLE  183 

the  carbon  cycle  and  rendering  the  carbon-dioxid  content  of  the 
atmosphere  nearly  constant. 

The  Nitrogen  Cycle.— Since  nitrogen  occurs  as  an  essential  part  of 
the  structure  of  every  plant  and  animal,  it  is  found  in  all  crops  and 
crop  residues.  It  occurs  in  the  top  soil  in  proteins,  protein  decom- 
position products,  ammonia,  nitrites,  and  nitrates.  It  is  not  found 
in  the  mineral  matter  of  the  earth  except  in  shales  and  other  deposits 
containing  the  residues  of  plant  and  animal  bodies.  Hence,  the 
quantity  in  the  combined  form  is  not  great  when  compared  with 
other  essential  elements.  Yet  it  is  required  by  all  living  organisms 
in  large  quantities.  Many  of  these  are  returning  it  to  its  inert 
atmospheric  form.  This  fact  led  Sir  William  Crooks,  in  his  famous 
address  before  the  British  Association  for  the  Advancement  of 
Science  in  1898,  to  predict  dire  calamity  to  the  human  race  if  science 
were  not  able  to  utilize  atmospheric  nitrogen. 

In  the  free  form,  nitrogen  occurs  in  enormous  quantities;  four- 
fifths  of  the  atmosphere  is  composed  of  it.  Dr.  Hopkins  has  pointed 
out  that  the  total  supply  of  nitrogen  over  each  acre  of  the  earth's 
surface,  if  available,  would  meet  the  needs  of  a  hundred-bushel  crop 
of  corn  every  year  for  500,000  years,  whereas  the  supply  of  carbon 
is  sufficient  for  such  crops  for  only  twro  years.  Nevertheless, 
carbon  has  no  commercial  value  as  plant-food,  while  nitrogen  in 
available  form  is  worth  from  15  to  20  cents  a  pound  on  the  market. 

The  same  atom  of  nitrogen  at  different  times  plays  many  different 
roles.  One  of  the  triumphs  of  agricultural  bacteriology  is  the 
advancement  which  it  has  made  in  following  nitrogen  through  its 
cycle. 

Nitrogen  occurs  in  the  plant  and  animal  mainly  in  the  form  of 
protein.  The  plant  protein  may  be  eaten  by  the  animal  and  produce 
animal  protein.  Either  may  reach  the  soil  and  decay.  The  nitro- 
gen eaten  by  animals  may  be  deposited  as  tissues  of  the  animal  or 
excreted  as  urea,  hippuric  or  uric  acid.  These  products  are  acted 
upon  by  bacteria  with  the  formation  of  ammonia. 

Either  the  plant  or  animal  proteins  may  reach  the  soil  where 
decay  sets  in  with  the  formation  of  albumoses,  proteoses,  peptones, 
peptids,  and  amino-acids.  The  amino-acids  are  then  deaminized 
with  the  formation  of  an  acid  and  ammonia.  The  process  is  spoken 
of  as  amrnonification. 

The  ammonia  does  not  accumulate  in  the  soil,  but  is  acted  upon 
by  other  bacteria,  the  nitrosomonas,  with  the  formation  of  nitrous 
acid.  This  is  quickly  taken  up  by  the  nitrobacter  and  oxidized 
to  nitric  acid  which  reacts  with  bases  in  the  soil  with  the  formation 
of  nitrates.  The  nitrates  are  the  main  source  of  nitrogen  for  the 
plants  which  build  from  them  and  carbon  dioxid,  amino-acids, 
peptids,  peptones,  proteoses,  albumoses,  and  finally  plant  proteins— 
and  the  nitrogen  has  completed  its  cycle.  If  this  were  the  whole 


184     THE  CARBON,  NITROGEN,  AND  PHOSPHORUS  CYCLES 

story  the  quantity  of  combined  nitrogen  in  the  world  would  remain 
constant.  But  it  is  not— there  are  many  leaks  in  the  cycle.  Some 
of  the  plants  and  animals  may  be  burned  with  the  liberation  of  free 
nitrogen.  Millions  of  pounds  of  it  reach  sewers,  and  from  here 
rivers,  lakes,  and  oceans.  In  time  this  is  broken  down  and  the 
nitrates  so  formed  are  reduced  by  denitrifying  bacteria  with  the 
liberation  of  gaseous  nitrogen.  The  processes  of  decay  continually 
going  on  may  also  liberate  free  nitrogen.  Furthermore,  millions  of 
pounds  of  nitrogen  are  returned  to  the  air  by  explosives.  So  the 
combined  nitrogen  would  continue  to  grow  less  were  it  not  that  other 
factors  are  at  work  in  nature  causing  it  to  combine.  Every  flash 
of  lightning  causes  some  nitrogen  to  combine  as  oxids,  but  the 
quantity  of  combined  nitrogen  thus  formed  is  relatively  insignificant. 
The  major  factors  are  biological.  There  are  within  the  soil  two 
great  groups  of  bacteria  which  possess  the  power  of  fixing  nitrogen. 
The  first— the  non-symbiotic  nitrogen-fixing  organisms  living  free 
in  the  soil— are  able,  with  the  energy  they  obtain  from  the  oxidation 
of  organic  carbon,  to  build  up  complex  organic  nitrogen  compounds. 
There  are  two  groups  of  these  organisms— the  aerobic  and  the  anae- 
robic, the  first  being  the  more  important.  The  other  class  of 
nitrogen-fixers  is  the  symbiotic;  these  live  in  conjunction  with 
legumes  and  obtain  from  them  carbonaceous  material,  and  in  return 
give  combined  nitrogen.  In  either  case  the  combined  nitrogen 
becomes  available  for  higher  plants.  Then  it  again  starts. on  its 
journey  through  the  living  and  the  dead. 

The  Sulphur  Cycle.— Sulphur  is  an  essential  element  for  all  plants  and 
animals,  but  the  quantity  required  for  normal  growth  and  develop- 
ment is  relatively  small  even  when  compared  with  the  small  per- 
centage found  in  soil.  It  occurs  in  the  soil  as  organic  and  inorganic 
sulphur.  The  former  is  derived  from  the  plant  and  animal  residues. 
These  are  acted  upon  by  microorganisms  with  the  liberation  of 
hydrogen  sulphid,  sulphur  dioxid,  and  sulphates.  Some  of  the  hydro- 
gen sulphid  is  carried  into  the  ocean  or  soil  by  the  first  rain;  some  of  it 
reacts  upon  the  iron  silicates  of  the  soil  and  forms  pyrite  or  marca- 
site,  but  most  of  it  is  oxidized  by  bacteria  with  the  formation  of 
sulphates.  The  sulphur  dioxid  is  also  further  oxidized  to  sulphates, 
when  they  are  again  taken  up  by  plants  and  start  anew  upon  their 
wonderful  journey  through  bacteria,  higher  plants,  and  animals. 

The  Phosphorus  Cycle.— Phosphorus  occurs  in  the  soil  in  the  form 
of  calcium,  aluminum,  and  iron  phosphate,  also  as  organic  phos- 
phorus. It  is  also  found  in  places  as  huge  deposits  of  rock  phos- 
phate. It  is  an  integral  part  of  every  living  plant  and  animal  cell. 
In  these  it  occurs  in  two  forms— organic  and  inorganic.  The  organic 
phosphorus  occurs  in  the  nucleo-proteins,  phospho-proteins,  and 
phospho-lipins. 

The  mineral  phosphates  of  the  soil  are  rendered  soluble  through 


THE  PHOSPHORUS  CYCLE  185 

bacterial  activity,  as  outlined  in  a  preceding  chapter.  This  is  taken 
up  by  the  living  plant  and  deposited  either  as  organic  or  inorganic 
phosphorus  compounds  within  the  plant  tissues.  The  plant  tissues, 
if  eaten  by  animals,  yield  phosphorus  to  the  animal  to  be  laid  down 
in  the  body  of  the  animal  as  organic  or  inorganic  compounds.  The 
excreta  of  animals  always  contain  phosphorus  in  both  organic  and 
inorganic  forms.  The  inorganic  phosphorus  is  readily  utilized  by 
plants  and  again  starts  on  its  cycle.  However,  the  organic  and 
animal  residues  must  be  mineralized  by  bacteria  before  they  can  be 
utilized  again  by  plants.  Microorganisms  split  off  the  carbonaceous 
material  and  the  phosphorus  is  liberated  mainly  in  the  form  of 
phosphates.  Under  some  conditions  mold  action  may  give  rise  to 
small  quantities  of  phosphin  which  must  be  again  oxidized  before 
being  available  to  higher  plants.  In  either  event,  the  resulting 
phosphate  is  now  ready  to  start  on  its  cyclic  journey  through  the 
plant  and  animal  organism.  This  is  dramatically  outlined  for  a 
phosphorus  atom  by  one  writer  as  follows : 

1  "Where  was  I  born?  Ah,  that  I  cannot  tell  you.  It  was  far, 
far  away  from  here,  deep  in  the  endless  abyss  of  space,  at  an  epoch 
so  distant  that  even  the  earth  on  which  you  live  had  not  been  formed 
as  yet;  not  even  the  great  sun,  now  blazing  in  his  glory,  nor  any  of 
the  innumerable  multitudes  of  stars  of  the  great  universe  now 
shining  in  the  sky,  had  as  yet  come  into  being.  No,  they  were  mere 
cold  whiffs  of  invisible  vapor,  scattered  over  all  space,  remnants  of 
worlds  vanished  seons  before  this  great  universe  began.  Out  of  the 
vast  I  came,  born  into  that  great  sea  of  ether  which  stretches 
unbroken  from  star  to  star  through  all  the  endless  depths  of  space. 
Some  vast  change,  some  murmuring  and  stirring  of  gigantic  forces 
in  its  bosom,  forces  .scarce  known,  scarce  dreamt  of,  but  working 
there  in  irresistible  might,  first  brought  me  into  being,  and  I  hung 
suspended  in  the  great  void.  It  was  utterly  cold  and  utterly  dark, 
and  gleaming  afar  in  the  distance  I  could  see  the  myriad  fires  of  the 
great  worlds  and  suns  of  space  shining  at  me  through  the  darkness. 
How  long  I  hung  in  the  void  I  know  not.  It  was  millions  upon 
millions  of  years.  Then  atoms  began  to  gather  round  me,  stream- 
wise,  coming  from  afar  in  phosphorescing  torrents,  and  I  perceived 
that  I  already  formed  part  of  a  mighty  mass  of  gas,  a  huge  nebula, 
which  stretched  its  gigantic  arms  out  for  millions  of  miles,  like  vast 
flaming  swords,  through  the  darkness  of  space.  And  so  I  hung  for 
seons  of  time,  while  atom  after  atom  in  an  endless  stream  flashed 
past  me  in  the  gloom,  while  the  great  nebula  slowly  drew  together 
in  its  glory,  and  began  to  take  shape  and  form.  Then  the  tempera- 
ture began  to  rise  in  leaps  and  bounds,  it  grew  stifling  hot,  and  great 
lightnings  flashed  and  quivered  about  me,  and  we  atoms  crowded 
more  and  more  together,  colliding,  whirling,  flying.  Each  second  I 
smote  a  thousand  million  atoms  and  at  each  collison  my  motion  grew 


ISC)     THE  CARBON,  NITROGEN,  AND  PHOSPHORUS  CYCLES 

more  and  more  violent,  until  after  millions  upon  millions  of  years  of 
this  tumult,  I  found  myself  part  of  an  immensely  hot  flaming  mass 
of  gas,  part  of  an  embryo  sun.  There  in  the  whirl  and  roar  of  this 
elemental  flame  I  remained  for  unthinkable  ages,  but  at  last  vast 
thunders  beneath  and  around  me  made  me  aware  that  something 
tremendous  was  happening.  It  was  a  world— my  first  world— 
gradually  condensing  out  of  the  fire  mist,  and  the  gigantic  explosions 
which  occurred  from  time  to  time  were  just  great  seas  of  boiling  rock 
leaping  upwards.  I  will  spare  you  the  account  of  how  I  entered 
into  that  world,  and  saw  it  slowly  form  and  develop  into  a  fair  planet, 
covered  with  wonderful  swarming  masses  of  living  creatures,  with 
great  cities  filled  with  busy  life,  and  wonderful  civilizations.  Nor 
will  I  tell  you  of  how  that  world  grew  old,  and  passed  into  a  vast 
desert,  and  finally,  after  wandering  for  aeons  of  time  in  darkness 
and  silence,  burst  suddenly  forth  into  flame,  the  victim  of  a  great 
cosmical  catastrophe,  and,  like  a  bubble,  vanished,  exploding  into 
incandescent  gas.  Nor  will  I  tell  you  of  how,  far  flung,  I  fell  upon 
another  world,  and  saw  this  world  too  in  time  perish;  and  of  how 
I  passed  from  world  to  world,  and  formed  part  of  world  after  world, 
wandering  in  mighty  migrations  through  space,  until  at  last  I  joined 
the  fire  mist  from  out  of  which,  ultimately,  this  present  world  of 
yours  condensed  amidst  titanic  convulsions.  You  will,  therefore, 
see  that  even  before  your  world  began,  I  was  old,  immensely  old. 
I  will  pass  over  all  this  and  come  to  a  time  quite  recent,  when  I 
found  myself  forming  part  of  the  molten  fire  underground.  Here  I 
lay  for  age  after  age,  while  the  land  above  me  was  being  eaten  away 
by  wind  and  rain  and  storm,  and  was  buried— continent  after 
continent  crumbling  into  ruin— into  the  great  ocean  waiting 
patiently  to  receive  it.  Now  I  was  urged  upward  by  vast  forces, 
slowly,  steadily,  for  thousands  of  years,  until  I  finally  was  uplifted 
to  form  part  of  a  hard,  cold  rock,  which  soon  reared  itself  into  a 
mighty  cliff,  beaten  upon  by  wind  and  rain  and  storm ;  I  have  a  dim 
recollection  of  looking  out  from  the  cliff  face  upon  a  widespread  blue 
sea,  filled  with  strange  vast  monsters,  which  have  long  since  vanished 
from  the  earth.  But  at  last  the  cliff  was  washed  away  and  I  passed 
into  the  great  body  of  the  sea,  and  was  absorbed  into  a  tiny  plant, 
living  beneath  the  salt  waters;  but  this  was  devoured  by  a  glittering 
gorgeous  fish,  and  so  I  entered  his  body.  Then  this  fish  was 
devoured  by  a  reptile,  which,  creeping  out  of  the  water,  entered  a 
swamp  and  died,  and  its  huge  body  decaying,  I  was  washed  into  the 
soil,  and  there  meeting  with  the  rootlet  of  a  plant,  I  entered  into  and 
formed  part  of  it;  and  this  was  eaten  by  an  animal;  and  so  I  entered 
into  its  body  and  formed  part  of  it;  and  this  was  eaten  by  an  animal; 
and  so  I  entered  into  its  body  and  formed  part  of  his  bones.  While 
we  were  crossing  a  ravine  one  bright  sunshiny  day,  millions  of  years 
ago,  a  green  monster  flashed  out  upon  us  and  slew  my  master  and 


THE  PHOSPHORUS  CYCLE  18? 

% 

devoured  me.  After  a  time  my  new  host  was  also  slain  in  a  similar 
manner,  and  his  body,  decaying  in  the  rank  grass  and  vegetation  of 
the  swamp,  I  was  ultimately  washed  out  to  sea  in  a  sudden  flood, 
which,  coming  down  from  the  hills,  swept  me  away.  Here  I 
mingled  with  the  mud  at  the  bottom  of  the  sea,  and  stayed  there  for 
millions  of  years,  and  became  covered  over  with  mighty  layers  of 
mud  and  sand,  and  sank  ever  deeper  and  deeper  into  the  earth,  and 
at  last  once  more  felt  the  glow  of  the  nether  fires.  Here  in  the  great 
gleaming  furnaces  of  the  deep  I  remained  for  many  millions  of  years, 
while  miles  above  me  the  world  changed  and  developed,  mountains 
came  and  went,  new  and  strange  creatures  evolved,  developed, 
filled  all  the  earth,  and  died  out  again.  One  day,  I  was  hurled  forth 
amidst  vast  thunderings  through  the  throat  of  a  great  volcano,  and 
formed  part  of  a  molten  lava  stream,  which  in  time  became  a  fertile 
field  covered  with  waving  crops  and  golden  grain.  Then  I  entered 
into  a  grain  of  corn,  and  wras  devoured  by  a  man  living  thousands  of 
years  ago,  a  mere  savage  you  would  term  him,  wild  and  fierce.  From 
him  I  passed  to  earth  once  more,  and  since  then  have  been  passing 
in  a  ceaseless  round  of  change  through  the  bodies  of  living  creatures. 
I  have  flown  through  the  air  in  a  bird,  I  have  swum  in  the  sea  in  a 
fish,  I  have  roamed  over  the  earth  in  a  beast,  I  have  formed  part  of 
innumerable  plants.  But  the  full  tale  would  only  weary  you, 
wonderful  as  it  is.  One  day,  a  few  years  ago,  I  was  devoured  by  an 
ox  while  forming  part  of  a  piece  of  grass,  and  soon  by  the  mysterious 
chemical  forces  of  its  body  I  was  made  to  form  part  of  its  bone. 
The  great  beast  was  slaughtered  by  men,  and  his  flesh  eaten,  and 
his  bones  burnt  to  a  fine  white  dust  in  a  furnace.  Out  of  this  dust, 
I,  the  tiny  phosphorus  atom,  was  distilled  in  a  furnace  and  found 
my  way  to  a  match  factory,  and  am  now  in  this  little  match-box 
lying  in  the  table  before  you.  Is  my  journey  finished?  Oh  dear  no, 
far  from  it.  I  shall  go  on  changing  and  journeying  and  dancing, 
age  after  age,,  even  until  the  world  fades  away  like  a  mist,  and  long 
after  all  that  you  see  and  hear  around  you  has  crumbled  away  and 
vanished  into  the  awful  maw  of  time.  I  have  been  taking  part  in 
the  great  dance  of  atoms  which  forms  the  basis  of  all  passing  things 
and  events,  for  millions  upon  millions  of  years,  and  shall  continue 
to  do  so  for  millions  and  millions  of  years  to  come.  I  may,  indeed, 
see  this  world  perish,  and  may  yet  dance  in  worlds  as  yet  unborn. 
My  future  will  be  probably  even  more  strange  than  my  past." 

REFERENCES. 

Kossowicz,  Alex.:     " Agrikulturmykologie,  I  Bodenbakteriologie'." 
Lafar,  Franz:     "Handbuch  der  Technischen  Mykologie,"  Dritter  Band. 


CHAPTER  XIX. 
PUTREFACTION,  FERMENTATION,  AND  DECAY. 

PUTREFACTION,  fermentation,  and  decay  are  in  reality  terms  which 
are  essentially  distinct  although  they  have  been  greatly  confused 
and  used  synonymously  even  by  professional  men.  As  pointed  out 
by  Kendall,  this  confusion  is  attributed  partly  to  the  use  of  terms 
to  designate  certain  processes  which  occur  in  nature*  before  these 
changes  were  studied  either  biologically  or  chemically. 

Definitions.— Fischer  considers  the  term  fermentation,  as  it  should 
be  used  in  bacteriology,  as  the  biochemical  decomposition  of  nitro- 
gen-free compounds,  chiefly  carbohydrates,  due  to  the  action  of 
microorganisms,  and  putrefaction  as  the  biochemical  decomposition 
of  nitrogenous  organic  compounds  by  the  action  of  microorganisms. 

The  distinction  which  is  usually  drawn  between  decay  and  putre- 
faction—as the  decomposition  of  nitrogenous  organic  substances  in 
the  presence  of  oxygen  on  the  one  hand,  and  the  absence  of  oxygen 
(or  with  a  limited  supply)  on  the  other— is  not  always  sharply 
defined.  The  end  products  in  both  cases  may  be  quite  similar. 
Nencki  found  that  in  the  decomposition  of  gelatin  at  40°  C.  in  the 
presence  of  air,  there  were  formed  in  four  days  for  every  100  parts 
of  the  original  substance  9.48  parts  of  ammonia,  24.2  parts  of 
volatile  fatty  acids,  12.2  parts  of  glycocol,  19.14  parts  of  peptone, 
and  6.45  parts  of  carbon  dioxid,  the  other  28.53  parts  being  unde- 
termined. Jeannert  repeated  these  experiments  with  the  exclusion 
of  air  and  found  as  the  decomposition  products  of  gelatin,  carbon 
dioxid,  ammonia,  a  gas  smelling  like  carbon  bisulphid,  acetic,  butyric, 
and  valeric  acids,  glycocol,  leucin,  and  a  colloidin  base-like  substance. 
He  concluded  from  these  and  other  experiments  that  (1)  the  decom- 
position of  nitrogenous  substances  and  of  carbohydrates  may  be 
accomplished  with  access  or  exclusion  of  air;  (2)  in  the  latter  case 
the  decomposition  is  considerably  less  rapid,  and  complete  decompo- 
sition requires  a  period  six  times  as  long;  and  (3)  the  more  simple 
chemical  products  formed  are  in  the  two  cases  identical. 

Nor  is  it  a  safe  criterion  to  state  that  putrefaction  is  accompanied 
by  the  formation  of  ill-smelling  substances,  for  this  is  usually  a 
quantitative  and  not  a  qualitative  difference.  Moreover,  Hirschler 
has  pointed  out  that  the  putrefaction  of  protein  substances  is  modi- 
fied by  the  presence  of  carbohydrates.  The  addition  of  various 
carbohydrates,  glycerin,  and  calcium  carbonate  changed  the  decom- 


ACTIVE  AGENTS  189 

position  of  meat  so  that  aromatic  products  of  putrefaction  could  not 
be  detected.  From  this  he  drew  the  conclusion  that  the  decomposi- 
tion of  protein  substances  in  the  presence  of  cane  sugar,  starch, 
dextrin,  glycerin,  or  lactic  acid  may  not  be  accompanied  by  the 
formation  of  the  characteristic  putrefaction  products—  indol,  phenol, 
and  oxyacids.  Nevertheless,  there  is  a  marked  quantitative  dif- 
ference in  the  two  processes—  decay  and  putrefaction.  The  former 
is  marked  by  the  volatilization  of  the  organic  constituents—  either 
protein  or  non-protein—while  the  non-  volatile  mineral  constituents 
are  left  behind  in  a  form  largely  available.  Putrefaction  is  the  rapid 
and  intense  decomposition  of  nitrogenous"  (for  the  most  part  protein) 
bodies  by  certain  bacteria,  usually  with  the  formation  of  large 
quantities  of  gaseous,  ill-smelling  products.  There  may  result  as 
intermediate  products,  basic  substances  often  having  highly  toxic 
properties.  These  substances  have  been  named  ptomaines  by 
Brieger.  Many  of  them  contain  only  carbon,  hydrogen,  and 
nitrogen,  and  are  ammonia-substitution  products.  Some  of  the 
simpler  ones  are  : 

Methylamin      .....      .      .      ......  (CH3)NH2 

Dimethylamin        .      .      ..........  (CH3)2NH 

Trimethylamin       ............  (CH3)3N 

Putrescin     .......     .  .......  NH2(CH2)4NH2 

Cadaverin   ..............  NH2(CH2)5NH2 

They  are  usually  protein-cleavage  products,  sometimes  resulting 
from  the  mere  removal  of  carbon  dioxid  from  the  carboxyl  of  the 
amino-acid.  Putrescin  may  be  formed  from  ornithin  thus: 


CH2—  CH2—  CH2—  CH—  COOH 

|  =|  +     C02 

NH2  NH2  NH2  NH2 

Ornithin  Putrescin 

and  cadaverin  from  lysin  : 

GHz—  GHz—  GHz—  OH;:—  CH—  COOH  CH;!—  CHr-  CH?—  CH2—  CH2 

I  i  I  I     +  co2 

NH2  NH2  NH2  NH2 

Lysin  Cadaverin 

In  putrefying  mixtures  the  ptomaines  appear  on  or  about  the 
fifth  or  seventh  day  after  putrefaction  sets  in,  and  disappear,  by 
further  cleavage,  more  or  less  rapidly,  yielding  less  complex  nitrog- 
enous substances  that  are  non-toxic. 

Active  Agents.—  Liebig  and  the  early  workers  considered  these 
changes  to  be  purely  chemical  processes.  The  ferment  was  to  them 
an  extremely  alterable  organic  substance  which  decomposed,  and 
by  decomposing  set  in  motion  its  own  elements.  The  momentum 
thus  engendered  is  sufficient  to  tear  to  pieces  the  fermenting  sub- 
stance. •  This  in  turn  then  possesses  the  power  of  imparting  to  other 
compounds  this  same  property,  or,  in  other  words,  they  considered 


190         PUTREFACTION,  FERMENTATION,  AND  DECAY 

fermentation  a  true  chemical  process.  This  was  overthrown  by 
Pasteur,  who  proved  fermentation  to  be  due  to  living  microscopic 
organisms,  and  it  came  to  be  generally  believed  that  putrefaction 
was  due  to  a  certain  microorganism—  Bacterium  termo.  Cohn 
wrote  in  1872  that  through  his  own  experiments,  as  well  as  through 
those  of  other  investigators,  he  was  convinced  that  Bad.  termo  was 
the  ferment  of  putrefaction  in  the  same  wray  that  yeast  is  the  alcoholic 
ferment.  He  considered  that  other  bacteria  may  play  a  secondary 
role,  but  that  Bad.  termo  is  the  primary  cause  of  putrefaction.  How- 
ever, bacteriologists  soon  came  to  realize  that  Bad.  termo  was  only 
a  general  name  given  to  the  many  species  of  rod-shaped  organisms 
occurring  in  decaying  substances.  In  1884  and  1885  Hauser 
isolated  three  distinct  species  of  bacteria  capable  of  causing  putre- 
faction— Proteus  vulgaris  (B.  proteus,  B.  vulgaris,  B.  zopfi),  Proteus 
mirabilis,  and  Proteus  zenkeri.  The  first  two  are  capable  of  liquefy- 
ing gelatin,  while  the  last  is  not.  Many  different  bacteria  are 
encountered  in  a  spontaneously  putrefying  substance.  Among  the 
most  active  which  have  been  studied  are,  according  to  Effront: 
the  family  of  Proteus,  B.  putrificus  coli  (Bienstock),  B.  perfringens 
(Veillon  and  Zuber),  Micrococcus  flavus  liquefaciens  (Fluegge), 
B.  gracilis  putidus  (Tissier  and  Martelly),  B.  bifermentans  sporo- 
genes  B.  diplococcus  griseus  non-li^uefaciens  (Tissier  and  Martelly), 
B.  coli  communis  (Escherich),  Streptococcus  pyogenes  (Doleris  and 
Pasteur),  and  Staphylococcus  pyogenes  albus  (Rosenbach).  These 
bacteria  are  very  widely  distributed,  B.  proteus  being  especially  apt 
to  occur  in  substances  undergoing  decomposition.  Its  presence  is 
constant  in  rotten  meat,  is  very  frequent  in  manure,  and  is  met  with 
in  large  numbers  even  in  normal  dejecta.  The  putrefying  bacteria 
are  usually  anaerobic,  but  there  are  often  very  active  aerobes. 

H.  Martelly  made  a  careful  study  of  the  bacterial  flora  of  putrefy- 
ing material  and  found  that  it  changed  from  period  to  period.  He 
found  at  first  Micrococcus  flavus,  Staphylococcus  albus,  B.  coli,  and 
Diplococcus  griseus.  Then  at  the  end  of  three  or  four  days  B. 
perfringens,  B.  sporogenes  appeared,  at  the  end  of  eight  to  ten  days 
he  detected  the  presence  of  B.  putidus,  B.  putrificus,  and  Proteus 
zenkeri,  and  after  three  months  there  remained  only  B.  putrificus, 
B.  putidus,  and  Diplococcus  griseus. 

Products  of  Putrefaction  and  Decay.— Due  to  the  trypsin  and 
erepsin  secreted  by  the  bacteria  the  proteins  are  broken  into  albu- 
moses,  peptones,  proteoses,  and  amino-acids,  and  even  in  very 
advanced  putrefaction  nitrogenous  substances  are  always  found 
which  give  the  protein  reactions.  The  amidases  secreted  by 
bacteria  give  rise  to  volatile  acids,  amins,  phenol  and  indol  deriva- 
tives. Effront  summarizes  the  products  formed  as  follows:  (1) 
Ammonia  and  amins— ethylamin,  propylamin,  and  trimethylamin; 
(2)  volatile  acids,  comprising  all  the  members  of  the  fatty  series  up 
to  caproic  acid;  (3)  aromatic  a.ciqls  and  oxyacids,  like  phenylpro- 


ACTIVE  AGENTS  191 

pionic,  oxypheny  lace  tic,  and  oxyphenylpropionic  acids;  (4)  phenol, 
indol,  skatol,  pyrrol,  and  its  derivatives,  these  bodies  sometimes 
being  in  very  small  quantities  or  even  completely  absent;  (5)  sulphur 
derivatives  like  methyl-mercaptan;  (6)  various  amino-acids,  leucin, 
tyrosin,  tryptophan,  and  ''sometimes  glycin,  creatinin,  etc. ;  (7) 
various  ptomains,  like  putrescin  and  cadaverin,  the  guanidins, 
cholin,  and  nurin,  pyridin,  hydrocolloidin,  etc. 

In  the  process  of  decay  the  carbon  and  hydrogen  is  liberated  as 
carbon  dioxid,  methane,  water,  and  other  volatile  products  with  the 
result  that  the  carbon  in  the  soil  tends  to  fall  off  relatively  to  the 
nitrogen  and  the  ratio  -§-,  which  in  the  original  plant  material 
is  about  40,  is  reduced  in  the  soil  to  10.  This  carbon-nitrogen  ratio 
varies  with  climatic  conditions,  also  with  soil  type  and  previous 
treatment.  Lawes  and  Gilbert,  as  quoted  by  Lipman,  give  the 
following  carbon-nitrogen  ratio  in  the  organic  matter  of  different 
soils : 

Cereal  roots  and  stubble 43.0 

Leguminous  stubble 23.0 

Dung 18.0 

Very  old  grassland 13.7 

Manitoba  prairie  soil 13.0 

Pasture  recently  laid  down .11.7 

Arable  soil '. 10.1 

Clay  subsoil 6.0 

Other  things  being  equal,  a  wide  carbon-nitrogen  ratio  indicates 
a  more  fertile  soil  than  a  narrow  carbon-nitrogen  ratio.  But 
this  must  always  be  interpreted  with  regard  to  the  climatic  condi- 
tion. In  the  arid  regions  the  carbon-nitrogen  ratio  is  narrow  when 
compared  with  soils  of  the  humid  regions,  yet  the  bacterial  activity 
of  the  former  is  just  as  active  as  that  of  the  latter. 

The  organic  substances  found  within  the  soil  are  called  humus  and 
result  from  the  action  of  bacteria  upon  the  plant  residues.  The 
composition  of  the  substance  varies  with  the  products  from  which 
it  has  been  formed,  also  the  degree  of  humification  which  has  taken 
place.  Moreover,  the  quantity  and  speed  with  which  humus  is 
formed  depends  upon  the  nature  and  condition  of  the  material  used 
and  the  physical,  chemical,  and  biological  conditions  of  the  soil. 
Hilgard  thinks  that  in  the  humid  regions  one  part  of  normal  soil 
humus  may  be  formed  from  five  to  six  parts  of  dry-plant  debris, 
whereas  in  the  arid  regions  from  eighteen  to  twenty  parts  of  the 
same  material  would  be  required.  Snyder  allowed  various  organic 
substances  to  humify  for  one  year  with  the  following  results: 

Per  cent, 
nitrogen 

1  part  fresh  cow      manure  yielded  33  parts  humus  containing      6.16 
1     "    green  clover        "  "       25      "  *  8.24 


1     "    meat  scraps         "  "11 

1      "    sawdust  "       10 

1     "    oat  straw  "  "         6 


10.96 
0.30 
2.50 


192         PUTREFACTION,  FERMENTATION,  AND  DECAY 

Humus  is  mainly  valuable  because  of  its  physical  effect  upon  the 
soil  and  because  of  its  content  of  nitrogen,  potassium,  and  phos- 
phorus which  are  slowly  liberated  by  bacteria.  The  beneficial  effect 
of  organic  matter  upon  the  bacterial  flora  of  the  soil  and  soil 
fertility,  however,  is  mainly  exerted  before  it  reaches  the  stage  of 
humus. 

Chemistry  of  the  Processes.— The  primary  and  secondary  products 
resulting  from  the  decay  of  organic  matter  in  the  soil  are  classed  as 
humus.  They  are  not,  as  was  once  believed,  a  few  comparatively 
simple  organic  compounds  but  are  a  heterogeneous  mixture  of 
colloidal  and  crystalline  organic  compounds  resulting  from  the 
action  of  bacteria  upon  plant  residues. 

The  chemical  composition  of  the  end  products  being  in  many 
cases  unknown,  the  chemistry  of  the  process  is  still  to  be  explained, 
but  we  have  some  very  suggestive  information  due  to  the  fact  that 
acids  and  alkalies  when  they  act  upon  carbohydrates  yield  brown 
humus-like  substances  very  similar  to,  if  not  identical  with,  the 
substances  found  in  the  soil  and  resulting  from  bacterial  activity. 

It  is  known  that  the  aldehyd  group  of  a  carbohydrate  easily 
opens  its  double  bonds  between  carbon  and  oxygen  and  adds  water 
to  form  a  polyhydric  alcohol,  as  follows : 

OH 
/ 

R— C=O     +     H2O  =  R— C\ 

|      OH 
H  H 

This  reacts  with  sodium  hydroxid  with  the  formation  of  the 
following  salt: 

H  H     ^  H        H 

+     NaOH     =     R— C &  +  •  H2O 

OH 

This  salt  is  unstable  and  the  molecule  forms  enols: 

HHHHHH  HHOH  OHH 

I       I       I       I      I        I  III  II 

— C— C— C— C— C— C— ONa   =  HO— C— C    =  C— C   =   C— C— ONa  +  2H2O 

I       I       I       I       I        I  I  I  I 

OH  OH  OH  OH  HO  HO  H  H  OH 

These  break  apart  at  the  double  bonds: 

H    H  OH  H  OH  H 

II  II 

HO— C— C      =    ;      =     C— C      =    ;      = 
I 

H 
H    H  OH  H  OH 

I       I  I       I                          I 

HO— C— C  =     C— G     -   ;     =     C— C 

I  I 

H  H 


CHEMISTRY  OF  THE  PROCESSES  193 

By  this  process  pieces  having  various  numbers  of  carbon  atoms 
are  formed,  all  of  which  are  very  reactive  in  their  nascent  state  due  to 
the  free  open  bonds  on  the  carbon  atom.  These  react  with  each 
other  and  give  rise  to  long  chain  compounds,  the  more  complex  of 
which  have  a  brown  color  arid  other  physical  and  chemical  charac- 
teristics of  soil  humus. 

Soil  humus  also  contains  nitrogen  which  would  come  through  the 
action  of  bacteria  upon  proteins.  The  products  resulting  through 
such  action  are  numerous  and  varied,  but  the  work  of  Schreiner  and 
his  associates  has  shown  them  to  be  the  following: 

Arginin  (CeHi4O2N4)  Nucleic  acid  (constituents  unknown) 

Adenin  (CsHsNs)  Oxalic  acid  (C2H2O4) 

Agroceric  acid  (C2iH42O3)  Picolin  carboxylic  acid  (CvHrC^N) 

Acrylic  acid  (C3H4O2)  Paraffinic  acid  (C24H48O2) 

Agrosteral  (C22H22OH2O)  Phytosterol  (C26H44O.H2O) 

Cytosin  (C4H5ON3H2O)  Pentosan  (C6H8O4) 

Cholin  (C5Hi5O2N)  Quanin  (CH8N8) 

Creatinin  (C4H7ON3)  Rhamose  (C6H14Oio) 

Creatin  (C4H9O2N3)  Succinic  acid  (CUHeC^) 
Dihydroxystearic  acid  (CisHseCh)         Saccharic  acid  (CeHsOio) 

Hentriacontan  (CsiHw)  Salicylic  aldehyd  (CeH4CHOOH) 

Histidin  (C6H9O2N3)  Trimethylamin  (C3H9N) 

Hypoxanthin  (C5H4ON4)  Trithiobenzaldehyd  (C6H5CSH)8 
Lignoceric  acid  (C24H48O2) 
Monohydroxystearic  acid  (Ci8H36O3) 
Mannite  (CeHuOe) 

REFERENCES. 

Vorhees,  Edward  B.  and  Lipman,  Jacob  G.:     "A  Review  of  Investigations  in  Soil 
Bacteriology,"  U.  S.  Dept.  Agr.,  Off.  Exp.  Sta.  Bui.  194. 
Fuller,    George   W. :     "Sewage   Disposal." 
Effront,  Prescott:     "Biochemical  Catalysts  in  Life  and  Industry." 


13 


CHAPTER  XX. 
AMMONIFICATION. 

IN  the  preceding  chapter  it  was  shown  that  one  of  the  final  prod- 
ucts resulting  from  putrefaction,  fermentation,  and  decay  is 
ammonia.  The  production  of  ammonia  through  the  intervention  of 
microorganisms  is  known  as  ammonification.  The  speed  with  which 
this  ammonia  is  formed  within  a  soil  varies  with  the  physical  and 
chemical  composition  of  the  soil  together  with  the  number  and 
physiological  efficiency  of  the  various  organisms  taking  part  in  the 
process. 

Although  it  has  been  known  for  some  time  that  small  quantities 
of  ammonia  occur  in  all  arable  soil,  its  formation  was  not  known 
to  be  due  to  a  biological  process  until  1893  when  Muntz  and  Coudon 
demonstrated  that  ammonia  is  no  longer  formed  in  soils  sterilized 
by  heat.  They,  together  with  Kayser,  isolated  from  soil  two  species 
of  Bacterium,  one  of  Bacillus,  two  of  Micrococcus,  and  two  of  molds 
—all  of  which  produced  ammonia  in  veal  bouillon,  and  all  but  one 
(a  micrococcus)  gave  the  same  results  in  soil.  From  these  results 
they  concluded  that  the  formation  of  ammonia  in  the  soil  is  the 
result  exclusively  of  the  conjoint  activity  of  numerous  lower  organ- 
isms of  very  widely  different  characters. 

This  conclusion  was  confirmed  the  same  year  by  Marchal  who 
isolated  from  the  soil  the  species  of  microorganisms  (molds,  yeast, 
and  bacteria)  which  were  the  most  prevalent,  and  determined  which 
of  these  had  the  power  of  transforming  nitrogenous  material  into 
ammonia.  Of  31  species  tested,  17  displayed  a  strong  ammonify- 
ing power.  Most  of  the  others  displayed  a  smaller  but  none  the 
less  distinct  ammonifying  power.  Molds  and  yeast  wrere  also  found 
to  produce  ammonia.  On  inoculation  into  a  solution  containing 
1.365  gms.  of  organic  matter  per  liter  the  various  organisms  were 
found  to  transform  the  following  proportion  of  nitrogen  into  am- 
monia in  twenty  days : 

B.  mycoides 46 

N  1 39 

Proteus  vulgaris 36 

B.  mesenterius  vulgatus 29 

Sarcina  lutea 27 

B.  janthinus 23 

B.  subtilis ' 19 

The  B.  mycoides  was  selected  by  him  for  special  investigation. 
This  organism  is  found  very  widely  distributed  in  nature.  It  is 


AMMONIF1CATION  195 

always  present  in  the  surface  layers  of  cultivated  soil,  and  is  found 
frequently  in  manure,  vegetable  mold,  composts,  and  in  the  humus 
of  forests.  It  occurs  at  times  in  air  and  natural  waters.  He  found 
that  when  inoculated  into  a  neutral  solution  of  albumen  the  medium 
soon  becomes  strongly  alkaline  due  to  the  accumulation  of  ammo- 
nium carbonate;  simultaneously  there  was  a  corresponding  decrease 
of  albumin.  The  analysis  of  the  atmosphere  in  which  the  culture 
was  confined  showed  a  marked  decrease  of  oxygen  with  a  corre- 
sponding increase  of  carbon  dioxid.  Hydrogen  and  nitrogen  were 
not  among  the  gaseous  products.  The  quantity  of  carbon  dioxid 
and  ammonia  formed  in  the  respiration  of  this  organism  were  nearly 
in  the  proportion  in  which  they  are  formed  in  the  complete  combus- 
tion of  albumin.  In  addition  to  these  two  substances,  there  were 
found  in  the  solution  small  quantities  of  peptones,  leucin,  tryosin, 
and  formic,  butyric  and  propionic  acids.  Marchal  considered  that 
in  the  life  processes  of  B.  mycoides  atmospheric  oxygen  is  made  to 
combine  with  the  constituents  of  albumin,  its  carbon  being  trans- 
formed into  carbon  dioxid,  its  sulphur  into  sulphuric  acid,  a  portion 
of  the  hydrogen  into  water,  the  ammonia  appearing  as  a  residual 
product.  He  assumed  the  following  equation: 

CwHmNwSOa     +     77O2      =     29H2O     +     72CO2     +     SO3     +     18NH3 

The  best  conditions  for  the  activity  of  the  organisms  are:  (1)  A 
temperature  of  about  30°  C.,  (2)  thorough  aeration,  (3)  a  slightly 
alkaline  medium,  and  (4)  a  dilute  solution  of  protein.  It  was  also 
found  that  this  organism  can  ammonify  not  only  albumin  but  also 
casein,  fibrin,  legumin,  glutin,  myosin,  serin,  peptones,  creatin. 
leucin,  tyrosin,  and  asparagin,  but  was  unable  to  utilize  urea,  urea 
nitrate,  or  ammonium  salts.  In  the  main  these  results  have  been 
amply  confirmed  by  a  great  number  of  investigators. 

C.  B.  Lipman  and  Burgess,  however,  have  demonstrated  that  B. 
mycoides  is  by  no  means  always  the  most  efficient  ammonifying 
bacterium,  for  even  this  organism  varies  greatly  in  its  activity, 
depending  upon  the  chemical  and  physical  conditions  of  the  sub- 
strata. They  make  the  following  critical  statements  concerning 
MarchaPs  findings:  "First,  the  results  of  solution  cultures  are  no 
criterion  as  to  the  results  to  be  obtained  in  soils.  .Secondly,  that 
no  two  forms  of  organic  nitrogen  are  attacked  and  ammonified  with 
the  same  vigor  by  any  one  organism.  Thirdly,  that  different  soils 
will  modify  an  organism's  power  to  ammonify  any  one  given  form 
of  nitrogen  very  markedly,  so  that  it  may  be  efficient  in  one  case  and 
feeble  in  another.  Fourthly,  that  the  ammonifying  efficiency  of 
organisms  is  greater  in  sandy  soil,  and  possibly  in  others,  than  in 
solutions,  for  we  have  obtained  a  transformation  of  41.98  per  cent, 
of  peptone  nitrogen  and  36.06  per  cent,  of  bat  guano  into  ammonia 


196 


AMMONIFICAT10N 


by  Sarcina  lutea  and  B.  mycoides,  respectively,  in  twelve  days  at 
temperatures  between  27°  C.  and  30°  C.,  while  Marchal  only 
obtained  similar  transformation  in  thirty  days  at  30°  C.  in  albumin 
solutions." 

Species  and  Distribution.— As  was  pointed  out  by  Marchal,  the 
ammonifying  organisms  are  very  widely  distributed  in  nature. 
The  power  to  split  off  ammonia  from  protein  is  a  characteristic  of 
the  majority  of  soil  bacteria.  Gage  noted  the  production  of 
ammonia  in  thirteen  out  of  twenty  cultures  of  sewage  bacteria  tested 


FIG.  27. — Ammonifying  bacteria. —  1.  Bacterium  mycoides;  X  3,000.  (Nadson.) 
2.  Bacterium  mycoides;  involution  forms;  X  3,000.  (Nadson.)  3.  Bacterium 
tumescens.  (Myec.)  4.  Proteus  vulgaris;  X  3,000.  (Nadson.)  5.  Proteus  vul- 
garis; involution  forms;  X  3,000.  (Nadson.)  (Lipman's  "Bacteria  in  Relation  to 
Country  Life.") 

by  him.  He  further  found  that  the  gelatin  liquefiers  have  an 
ammonifying  power  nearly  twice  as  great  as  the  non-liquefiers. 
Chester  found  all  but  one  of  the  organisms  tested  by  him  capable 
of  producing  ammonia.  C.  B.  Lipman  tested  the  following  fifteen 
organisms  in  soils:  B.  mesentericus  vulgatus,  Ps.  putida,  B.  vulgatus, 
B.  megatherium,  B.  mycoides,  B.  subtilis,  B.  tumescens,  Sarcina 
lutea,  B.  proteus  vulgaris,  B.  icteroides,  B.  ramosus,  Streptothrix, 
sp.,  Ps.  fluorescent,  B.  vulgaris  (navy  strain),  and  Mic.  tetragenus  as 
to  their  ammonifying  powers  of  dried  blood,  tankage,  cotton-seed 


AND  DISTRIBUTION 


197 


meal,  sheep  and  goat  manure,  peptone,  fish  guano,  and  bat  guano. 
He  found  that  while  all  produced  ammonia,  their  efficiency  varied 
greatly,  depending  upon  the  nature  of  the  soil  and  the  nitrogenous 
material  to  be  ammonified,  B.  tumescens,  however,  on  the  whole 
appeared  to  be  the  most  efficient  organism  tested. 


FIG.  28.—  Ammonifying  bacteria.—  1.  Proteus  rulgaris;  X  2,600.  (Rodella.) 
2.  Bacillus  megatherium;  X  2,600.  (Hinterberger.)  3.  Bacillus  my  coides;  X  2,600. 
(Emmerling.)  4.  Bacillus  cereus;  X  2,600.  (Wilhelmy.)  Lipman's  "Bacteria  in 
Relation  to  Country  Life." 


Moreover,  many  fungi,  as  shown  by  McLean  and  Wilson,  possess 
the  power  of  rapidly  ammonifying  cotton-seed  meal  and  dried  blood. 
Fungi  belonging  to  the  Moniliacece  were  more  active  ammonifiers 
than  were  members  of  the  AspergillacecB,  Mucoracece,  or  Dematiacece. 

An  idea  as  to  the  number  of  ammonifying  organisms  which  must 
occur  in  soils  may  be  gleaned  from  the  fact  that  Conn  found  about 
10  per  cent,  of  a  soil's  flora  to  be  rapid  liquefiers,  principally  Ps. 


198  AMMONIFICATION 

fluorescens,  and  many  others  are  slow  liquefiers.  This  indicates 
that  the  class  of  organisms  must  play  a  very  important  role  in  the 
degradation  of  the  nitrogenous  material  of  the  soil. 

It  is  quite  likely  that  the  organisms  are  even  more  efficient  in  the 
soil  in  the  mixed  cultures  than  they  are  in  the  pure  cultures.  For 
the  transforming  of  protein  nitrogen  to  ammonia  is  a  complex 
process  which  must  proceed  by  steps  and  some  organisms  must  be 
more  efficient  than  are  others  in  specific  phases  of  the  reaction. 
But  so  far  we  have  little  definite  information  on  this  subject. 

Methods.— Two  methods  are  in  general  use  for  the  determination 
of  the  ammonifying  powers  of  the  soil.  The  one  in  which  a  definite 
portion  of  soil  is  inoculated  into  a  liquid  media  and  after  a  given 
time  the  ammonia  determined;  in  the  other  the  nitrogenous  sub- 
stance is  incorporated  into  the  soil  and  after  a  definite  period  the 
ammonia  determined.  The  latter  method  would  appear  to  approach 
more  nearly  field  conditions,  but  both  methods  have  their  advocates. 
It  is  not  my  purpose  to  go  into  the  claims  made  for  each,  but  suffice 
it  to  state  that  Lohnis,  who  has  made  a  careful  study  of  each,  finds 
the  more  important  factors  in  both  to  be:  (1)  Nature  and  quantity 
of  material  used  as  substrata;  (2)  concentration  and  distribution  of 
the  substrata  in  the  medium;  (3)  aeration;  (4)  diffusion,  absorption, 
destruction  or  evaporation  of  metabolic  products;  (5)  reaction  of  the 
medium;  (6)  temperature;  and  (7)  duration  of  the  experiment. 

As  was  pointed  out  by  Pagnoul  in  1895,  the  formation  of  ammonia 
in  the  soil  is  only  a  transition  state  of  organic  nitrogen  in  passing 
to  the  nitrates.  So  that  with  either  the  solution  or  soil  method, 
what  we  measure  is  the  accumulation  of  ammonia  in  the  media  and 
not  the  actual  quantity  formed.  Various  factors  may  enter  and  slow 
down  the  quantity  of  ammonia  formed.  This  would  be  indicated 
by  a  smaller  quantity  of  ammonia  in  the  soil,  or  the  speed  with  which 
the  ammonia  is  transformed  into  nitrates  may  decrease,  and  hence  the 
ammonia  accumulates  while  the  actual  quantity  found  is  the  same. 
Moreover,  it  is  well  known  that  many  microorganisms  possess  the 
power  of  transforming  ammonia  into  protein  nitrogen,  and  this 
factor  may  either  increase  or  decrease  with  a  corresponding  change 
in  the  ammonia  of  the  soil.  Where  large  quantities  of  ammonia  are 
being  formed,  part  of  it  may  be  lost  from  the  medium  by  volatiliza- 
tion. The  extent  of  this  loss  varies  with  the  soil.  Lemmermann 
and  Fresenius  found  the  addition  of  calcium  carbonate  to  a  soil  to 
the  extent  of  1  per  cent,  reduced  the  volatilization  of  ammonium 
carbonate  and  increased  the  absorptive  power  of  the  soil  for  ammonia. 
Calcium  sulphate  and  chlorid  and  magnesium  chlorid  have  a  similar 
effect.  Caustic  lime  has  the  opposite  effect.  The  zeolites  are  very 
effective  in  reducing  the  loss  of  ammonia  from  soil,  and  according  to 
Pfeiffer  and  coworkers  the  nitrogen  so  fixed  is  so  firmly  held  that  it 
does  not  become  available  to  plants  during  the  first  season. 


METHODS  199 

Material  Ammonified.— The  speed  with  which  ammonia  is  formed 
within  a  soil  varies  greatly,  depending  upon  the  nature  of  the 
material  to  be  ammonified.  Lipman  and  his  associates  found  the 
following  proportions  of  nitrogen  were  transformed  into  ammonia 
in  six  days: 

Concentrated  tankage 56.66  per  cent. 

Ground  fish 47.16 

Cow  manure,  solid  and  liquid  excreta 32 . 60 

Dried  blood 16.74 

Bone  meal 16.65 

Cow  manure,  solid  excreta 5.39 

Cotton-seed  meal .      .      .      .  4.95 

However,  this  order  is  not  always  maintained,  for  C.  B.  Lipman 
has  found  it  to  vary  with  the  soil  and  with  the  bacterial  flora. 
Lipman  and  Brown  consider  the  carbon-nitrogen  ratio  important  in 
determining  the  rate  of  ammonification  of  nitrogenous  materials, 
and  then  the  modification  of  this  ratio  by  soluble  carbohydrates  or 
by  other  soluble  compounds  may  lead  to  changes  in  the  numbers 
and  species  of  the  microorganisms  in  the  soil  or  culture  solutions 
and  a  consequent  depressed  or  intensified  ammonification,  depend- 
ing on  the  character  of  the  nitrogenous  fertilizer. 

The  addition  of  dextrose,  sucrose,  lactose,  maltose,  and  mannite, 
according  to  Lipman  and  Brown,  decrease  the  accumulation  of 
ammonia  in  the  soil.  Kelley  found  that  by  adding  1.586  gms.  of 
starch  to  1.072  gms.  of  casein  the  quantity  of  ammonia  in  the  soil 
at  the  end  of  nine  days  was  decreased  50  per  cent. 

In  the  presence  of  the  carbohydrates  the  decrease  may  be  either 
real  or  apparent.  The  true  decrease  may  be  due  to  the  carbohydrate 
which  causes  the  organism  to  use  only  sufficient  protein  to  meet  its 
nitrogen  metabolism  when  only  a  small  quantity  of  ammonia  would 
accumulate. 

The  apparent  decrease  is  probably  due  to  an  acceleration  of  the 
speed  with  which  ammonia  is  transformed  into  protein  nitrogen. 
Inert  organic  substances  in  general,  such  as  starch,  cellulose,  and 
peat,  usually  decrease  the  speed  of  ammonification.  This  is  due, 
according  to  Rahn,  to  the  substance  making  some  of  the  soil  moisture 
unavailable  to  the  bacteria,  for  he  found  that  when  the  moisture  is 
sufficiently  great  cellulose  acts  as  a  stimulant  to  ammonification, 
probably  by  holding  the  sand  particles  farther  apart  and  thus  in- 
creasing aeration.  Dzierzbicki  has  found  that  small  amounts  of 
some  humic  acid  salts  increase  ammonification. 

The  addition  of  manure  to  a  soil  greatly  increases  the  ammonia 
produced  in  a  soil.  This  is  illustrated  by  the  following  results 
obtained  by  Greaves  and  Carter.  In  the  first  column  is  given  the 
per  cent,  of  ammonia  found,  the  untreated  soil  being  taken  as  100 
per  cent.  The  various  quantities  of  manure  were  applied  to  the 


200  AMMONIFICATION 

soil  in  pots  and  after  four  months  the  ammonifying  powers  deter- 
mined. In  the  second  and  third  columns  are  given  the  results  from 
actual  field  soil  receiving  each  year  the  designated  quantity  of 
manure. 

Pot  Field  Experiment 

Treatment  Experiment  Fallow  Cropped 

No  manure 100  100  100 

5  tons  of  manure 122  147  129 

10     "     "         "          140 

15     "     "         "          152  188  183 

20     "     "         "          160 

25     "     "         "          180 

Crops  grown  on  a  soil  decrease  its  ammonifying  powers.  In  the 
order  of  their  decreasing  ammonifying  powers  came  fallow,  potato, 
corn,  oats,  and  alfalfa. 

Influence  of  Soil  and  Climatic  Conditions.— Lipman  points  out 
that  the  ammonia  production  in  a  soil  is  affected  by  (1)  its  mechani- 
cal and  chemical  composition,  (2)  by  fertilizer  treatment,  and  (3) 
by  methods  of  tillage  and  cropping.  He  further  states  that  the 
mechanical  composition  of  the  soil  determines  the  proportion  of 
aerobic  and  anaerobic  organisms  in  a  soil.  If  the  latter  predominate, 
the  production  of  ammonia  is  comparatively  rapid.  Yet  Fischer 
reports  a  more  intense  ammonification  of  dried  blood  in  light  than 
in  heavy  soils,  but  under  this  case  it  is  possible  that  the  loss  of 
nitrogen  from  the  sandy  soil  was  sufficient  to  account  for  the 
observed  difference.  C.  B.  Lipman  has  shown  that  the  order  within 
various  soils  differs  with  the  various  ammonifying  organisms. 
Moreover,  he  and  Waynick  found  the  removal  of  California  soil  to 
Kansas  increased  the  ammonifying  powers  about  one-third,  but 
removal  to  Maryland  had  little  effect.  Whereas  Kansas  soil  re- 
moved to  California  loses  greatly  in  its  ammonifying  power,  yet 
Kansas  soil  transported  to  Maryland  suffers  little  change.  We  must, 
therefore,  conclude  that  climate  exerts  a  great  influence  on  the 
ammonifying  powers  of  the  soil. 

The  quantity  of  ammonia  in  a  soil  varies  from  season  to  season 
and  from  period  to  period.  Green  found  that  as  regards  the 
ammonification  of  the  organic  manures— flesh  meal,  horn  meal,  and 
blood  meal— the  bacterial  activities  rise  from  August  to  October, 
with  a  tendency  to  fall  or  remain  constant  in  November,  rising  to  a 
maximum  in  December.  This  was  followed  by  a  minimum  in 
February  and  a  low  maximum  in  April,  and  from  April  to  July  there 
was  a  slight  fall  which  probably  continued  to  a  summer  minimum 
commencing  in  August. 

Moisture.— The  influence  of  moisture  on  the  ammonia  formed  in 
the  soil  is  very  great.  Lipman  and  Brown  found  ammonification 
in  a  loam  soil  increased  with  increased  water  content  even  up  to 
35  per  cent,  of  the^eight  of  the  soil.  However,  later  they  and 


MOISTURE  201 

Owen  found  ammonification  to  increase  as  the  water  added  increased 
up  to  a  certain  percentage,  which  varies  with  the  physical  nature 
of  the  soil,  but  larger  quantities  of  water  reduced  the  ammonia 
recovered.  The  work  clearly  demonstrated  that  the  optimum 
moisture  content  for  maximum  ammonification  is  higher  than  it  is 
for  maximum  nitrification.  The  quantitative  difference  between 
the  two  processes  in  the  same  soil  was  found  by  Sharp.  Ammoni- 
fication was  most  rapid  with  a  25  per  cent,  moisture  content  and  was 
not  markedly  affected  by  3  per  cent,  differences.  Nitrification  was 
at  its  maximum  when  the  soil  contained  19  per  cent,  of  water.  When 
it  was  increased  to  25  per  cent.,  the  rate  of  nitrification  was  decreased 
50  per  cent. 

The  work  of  Greaves  and  Carter  shows  that  ammonification  is 
at  its  maximum  in  a  soil  containing  60  per  cent,  of  its  water-holding 
capacity,  as  is  illustrated  in  Fig.  29  which  gives  the  results  obtained 
with  twenty-two  soils.  Moreover,  according  to  Briggs  the  moisture- 
holding  capacity,  the  wilting  coefficient,  the  moisture  equivalent, 
and  the  hygroscopic  coefficient  are  related  by  linear  equations : 

C    =    2.9  W    +    21 

C      =     1.57E      +     21 
C      =     4.26H      +     21 

where  C  is  written  for  the  moisture  capacity  as  defined  by  Hilgard, 
W  for  wilting  coefficient,  E  for  moisture  equivalent,  and  H  for 
hygroscopic  coefficient.  Therefore,  taking  the  maximum  ammoni- 
cation  at  0.6  of  the  water-holding  capacity  we  could  relate  these 
other  soil  constants  to  maximum  ammonification  by  a  similar  set  of 
linear  equations.  Thus: 

Ma  =  .942E  +  12.6 
Ma  =  1.74  W  +  12.6 
Ma  =  2.55  H  +  12.6 

writing  Ma  for  per  cent,  water  for  maximum  ammonification. 

Variation  in  moisture  content  of  a  soil  changes  its  ammonifying 
flora,  for  soils  held  at  various  moisture  contents  for  several  months 
show  different  ammonifying  efficiency,  as  may  be  seen  from  the 
following  results  obtained  by  Greaves  and  Carter.  These  soils 
were  held  at  the  indicated  moisture  content  for  three  months  and 
then  all  brought  to  20  per  cent,  moisture  and  after  four  days  the 
ammonia  determined. 

Per  cent.  Ammonia 
Moisture  Added  Produced 

12.5  per  cent,  water 100 

15.0  per  cent,  water Ill 

17.5  per  cent,  water 113 

20.0  per  cent,  water 123 

22 . 5  per  cent,  water 119 


202 


AMMONIFICATION 


This  increased  aminonification  with  increased  moisture  content  is 
due,  according  to  Lipman,  to  the  suppression  of  the  aerobic  decay 
bacteria  and  an  acceleration  of  the  anaerobic  putrefactive  bacteria. 


§ 


• 


FIG.  29. — Average  percentages  of  ammonia  produced  in  soils  receiving  varying 
quantities  of  water.  The  quantity  produced  at  60  per  cent,  is  taken  as  100;  on  the 
ordinate  is  given  the  per  cent,  of  ammonia  formed  whereas  on  the  abscissa  is  given 
water  applied  as  per  cent,  of  water-holding  capacity. 

Aeration.— Carbone  considers  oxygen  essential  as  a  rule  to  the 
most  efficient  action  of  organisms  in  decomposing  organic  matter, 
and  he  points  out  that  it  is  not  possible  to  segregate  the  agents  of 
decomposition  strictly  into  aerobes  and  anaerobes.  The  aerobes 
are  the  more  active  agents  in  the  decomposition  of  carbonaceous 
material  with  the  formatjpn  of  humus.  But  Dzierzbicki  found  a 


PHOSPHORUS  203 

strong  aeration  decreased— at  least  in  many  cases— the  quantity  of 
ammonia  split  off  from  peptone  solutions  which  had  been  inoculated 
with  soil.  Plimmer,  working  with  a  Dunkirk  clay  loam,  failed  to 
find  any  optimum  oxygen-,content  for  the  maximum  production  of 
ammonia.  Under  purely  anaerobic  conditions,  caused  by  an 
atmosphere  of  pure  carbon  dioxid,  there  was  somewhat  less  ammonia 
produced  than  when  oxygen  was  present  at  the  beginning,  but  even 
under  these  conditions  ammonia  was  formed  in  rather  large  pro- 
portions. This  would  probably  vary  with  the  specific  bacterial 
flora  of  the  soil,  for  the  work  of  Marchal  demonstrated  that  the 
formation  of  ammonia  is  favored  by  the  unhindered  access  of  oxygen, 
and  in  the  process  considerable  quantities  of  oxygen  are  used  up  and 
a  nearly  corresponding  quantity  of  carbon  dioxid  produced.  There 
is  the  possibility  of  the  carbon  dioxid  resulting  from  side  reactions, 
the  oxidation  of  the  carbon  chain  compounds  which  have  been 
deaminized,  and  not  due  to  the  main  process  of  ammonification. 
For  theoretically,  at  least,  ammonification  can  be  considered  as  a 
true  hydrolytic  reaction.  The  microorganism,  however,  gets  its 
energy  from  the  oxidation  of  the  carbon,  and  where  conditions  are 
favorable  ammonia  production  follows  very  closely  the  evolution 
of  carbon  dioxid.  But  Gainey  found  unfavorable  conditions  to 
have  a  more  detrimental  effect  upon  the  f ormatiori  of  ammonia  than 
upon  the  production  of  carbon  dioxid. 

Lime  and  Magnesia.  —  These  exert  a  marked  effect  on  ammoni- 
fication, and  Vorhees,  Lipman,  and  Brown  found  magnesium  lime 
usually  superior  to  non-magnesium  lime  in  this  respect.  Its  effect 
varied,  depending  upon  the  character  of  the  organic  matter  to  be 
ammonified  and  the  crop  grown  upon  the  soil.  Lipman,  Brown, 
and  Owen  found  lime  carbonate  in  which  the  large  per  cent,  of 
boron  proved  to  be  the  factor  which  hindered  decay  bacteria. 

The  increased  ammonia  resulting  from  the  use  of  the  magnesium 
lime  may  be  due  to  an  apparent  and  not  to  a  real  increase  in  ammoni- 
fication. For  Fischer  has  noted  that  calcium  carbonate  increased 
the  speed  with  which  ammonia  sulphate  is  transformed  into  protein 
to  a  greater  extent  than  did  magnesium  carbonate.  Moreover, 
Lipman  and  Green  found  that  magnesium  carbonate  interferes  with 
the  speed  with  which  nitrite  is  converted  into  nitrate  which  would 
slow  down  the  action  of  the  nitrosomonas.  In  either  case  this 
would  increase  the  accumulation  of  ammonia  in  the  soil,  which  may 
be  interpreted  as  increased  ammonification. 

Phosphorus.— In  experiments  on  ammonia  cleavage  by  Dzierzbicki 
in  peptone  solutions,  it  was  found  that  the  intensity  of  such  cleavage 
depends  not  only  on  the  bacterial  flora  of  the  soil  but  more  so  on  its 
chemical  composition  and  especially  on  the  presence  of  phosphoric 
acid.  Monocalcium  and  dicalcium  phosphate  are  equally  effective, 


204  AMMONIFICA  TION 

according  to  Lipman,  in  stimulating  the  activities  of  the  decay 
bacteria.  Thomas  slag  increases  the  production  of  ammonia  to 
a  considerable  extent.  Where  acid  phosphate  is  applied  to  a  soil 
the  increased  ammonification  resulting,  according  to  McLean  and 
Wilson,  is  due  to  fungi  rather  than  to  bacteria,  but  this  would 
probably  vary  with  the  magnesium  and  calcium  carbonate  content 
of  the  soil. 

Chemistry  of  the  Process.— Rettger  and  his  associates  have  shown 
that  bacteria  are  unable  to  attack  or  bring  about  the  decomposition 
of  proteins  without  the  aid  of  enzymes  or  other  proteolytic  agents, 
whereas  Itano  using  the  formal  titration  method  of  Sorenson  has 
shown  that  B.  subtilis  produces  a  gradual  increase  of  formal-titrating 
nitrogen  for  a  period  of  two  hundred  and  forty  hours.  The  greatest 
proteolysis  took  place  toward  the  optimum  hydrogen-ion  concentra- 
tion. He,  therefore,  suggested  that  the  enzyme  is  probably  tryptic- 
like  in  nature,  and  endocellular.  Moreover,  as  was  seen  in  the  preced- 
ing chapter,  a  number  of  amino-acids  have  been  isolated  from  the  soil. 
Hence  it  is  most  natural  to  assume  that  the  disintegration  of  pro- 
teins in  the  soil  is  primarily  protein  hydrolysis  catalyzed  by  endo- 
or  exo-enzymes  secreted  by  the  decay  bacteria.  The  enzymes- 
pepsin,  trypsin,  erepsin,  and  probably  other  protoclastic  enzymes- 
are  capable  of  hydrolyzing  proteins  with  the  formation  of  some 
eighteen  amino-acids.  The  number  and  quantity  of  each  depends 
upon  the  specific  protein  hydrolyzed.  Incomplete  hydrolysis 
results  in  the  production  of  a  number  of  intermediate  substances 
variously  designated  in  the  order  of  decreasing  complexity— 
proteoses,  peptones,  and  polypeptids. 

Taylor  suggests  the  scheme  given  on  page  205  as  indicating  the 
stages  in  the  hydrolysis  of  the  protein  molecule. 

There  are  reasons  for  believing  that  in  the  process  of  ammonifica- 
tion the  hydrolysis  is  similar  to  this,  for  Marchal  showed  that  the 
ammonifiers  are  capable  of  hydrolyzing  proteins,  proteoses,  and 
peptones.  Moreover,  many  of  the  protein  hydrolytic  products  are 
found  in  the  soil,  and  Miyaka  has  shown  from  a  mathematical 
analysis  that  the  process  of  ammonification  is  an  autocatalytic 
chemical  reaction  in  which  the  increase  of  ammonia  in  the  process 
is  in  accordance  with  the  formula : 

Log-    x 

=     K(t-ti) 
A-x 

Where  x  is  the  amount  of  ammonia  which  has  been  produced  at 
time  (t),  a  is  the  total  amount  of  ammonia  produced  during  the 
process,  K  is  a  constant,  and  t\  is  the  time  at  which  half  of  the  total 
amount  of  ammonia  is  produced. 


CHEMISTRY  OF  THE  PROCESS 


205 


f  I 

•i  4 

•§  .S 
&S 


I 


1 


w  03    d 
»  f  J 

'•3-32 


206  AMMONIFICA  TION 

The  quantity  and  quality  of  the  products  formed  would  depend 
upon  the  specific  enzyme  or  enzymes  secreted  by  the  microorgan- 
isms. Where  pepsin  is  the  main  ferment  large  quantities  of  proteoses 
and  peptones  would  be  formed,  while  if  trypsin  is  the  active  agent 
the  ammo-acids  would  also  occur,  and  with  erepsin  the  amino-acids 
would  be  formed  even  more  rapidly.  It  is  not  unreasonable  to 
suppose  that  in  a  soil  having  a  high  ammonifying  efficiency  the 
ammonifying  flora  are  not  only  numerous  but  they  are  active 
secreters  of  proteoclastic  ferments.  Moreover,  this  disintegration 
of  the  protein  molecule  which  eventually  converts  nearly  the  whole 
of  the  organic  nitrogen  into  ammonia  results  from  the  combined 
action  of  numerous  microorganisms  of  different  species.  The 
products  elaborated  by  one  class  probably  serve  as  the  point  of 
attack  for  another. 

Potter  and  Snyder  found  there  was  no  tendency  for  the  amino- 
acids  to  accumulate  in  the  soil,  but  they  fluctuated  with  the  ammonia, 
thus  indicating  a  connection  between  the  two.  Jodidi  added 
various  amino-acids  and  acid  amids,  including  glycocol,  leucin, 
phenylalanin,  asparagin,  aspartic  acid,  glutanic  acid,  tyrosin, 
alanin,  cadaverin,  acetimid  and  propionamid  to  the  soil,  and  after 
from  two  to  ten  days  determined  the  ammonia,  although  the 
transformation  was  not  quantitative,  probably  due  to  other  reac- 
tions occurring  simultaneously.  Yet  it  was  evident  that  the 
amino-acids  and  acid  amids  examined  readily  undergo  in  the  soil 
the  process  of  ammonification,  and,  all  other  things  being  equal,  the 
rate  of  transformation  is  greatly  influenced  by  the  chemical  struct- 
ure so  that  amino-acids  and  acid  amids  of  equal  structure  yield 
about  the  same  proportion  of  ammonia. 

Effront  in  1905  demonstrated  that  there  are  produced  by  soil 
bacteria  amidases.  The  process  of  deamination  was  at  first 
thought  to  be  one  of  simple  hydrolysis,  as  follows : 

R  CH  NH2    -  -    COOH    +    HOH   =    R    -  -    CHOH    -  -    COOH    +    NH3 

But  Neubauer  and  Fromberz  concluded  that  the  primary  pathway 
of  deamination  within  the  animal  organism  was  oxidative  and  not 
hydroly  tic : 

R  "R  R 

|     H  OH 

2C/  +     O2      =     2C/  =     2C      =     O     +     2NH3 

|\NH2  |\NH2 

COOH  COOH  COOH 

Amino-acid  Oxy-amino-acid     Keto-acids 

The  amino-acids  containing  sulphur  would  probably  in  the 
presence  of  sufficient  oxygen  first  oxidize  the  sulphur  atom  forming  a 
derivative  of  sulphuric  acid: 

•CH-SH  CH2SO3H 

2CH.NH2     +     302 
CQOH  COOH 


CHEMISTRY  OF  THE  PROCESS  207 

And  this  would  be  deaminized : 

CH2SO3H  CH2SO3H  CH2SO3H 

I      H  |      OH  | 

2C/  +     O2     =     C/  =     2C      =     O     +     2NH3 
|\NH2                                |\NH2 
COOH                                 COOH  COOH 

In  any  case  the  resulting  ammonia  would  be  fixed  within  the  soil 
by  the  zeolites,  acid  radicals,  or  lost  through  volatilization.  That 
which  is  retained  in  the  soil  is  later  taken  up  by  plants  or  trans- 
formed by  bacteria  into  protein  or  nitrate  nitrogen. 

For  each  molecule  of  ammonia  formed  there  would  result  a  corre- 
sponding molecule  of  volatile  acid,  or  oxy-acid,  or  else  of  alcohol 
which  may  serve  as  a  source  of  carbon  for  other  microorganisms, 
possible  for  the  azofiers. 

REFERENCES. 

Lohnis,  F. :     "Handbuch  der  Landwirtschaftlichen  Bakteriologie." 
Lafar,  Franz:     "Handbuch  der  Technischen  Mykologie,"  Dritter  Band. 
Voorhees,  Edward,  B.,  and  Lipman,  J.  G.:     "Review  of  Investigations  in  Soil 
Bacteriology"  (U.S.D.A.  Off.  Exp.  Sta.  Bui.  194). 


CHAPTER  XXI. 
NITRIFICATION. 

THE  term  nitrification  refers  to  the  oxidation  of  ether  ammonia 
or  nitrites  to  nitrates.  It  is  often  used  in  a  broader  sense  to  imply 
the  production  of  nitrates  from  decomposing  organic  material. 
The  process  of  nitrification  was  made  use  of  in  the  manufacture  of 
saltpeter  to  supply  the  large  quantities  of  gunpowder  consumed 
in  the  almost  incessant  wars  of  Europe.  In  the  eighteen  century 
the  artificial  production  of  saltpeter  in  beds  of  decaying  organic 
matter  reached  a  high  degree  of  perfection.  Especially  was  this 
true  in  Sweden,  Switzerland,  and  France,  where  nitre  was  collected 
as  a  part  of  each  .farmer's  tax.  In  the  year  1777  the  French  Govern- 
ment issued  special  instructions  for  manufacture  of  saltpeter.  In 
these  there  was  given  special  attention  to  the  form  of  pit  to  be 
used,  the  covering  of  the  organic  matter,  the  arrangement  for  the 
free  entry  of  air,  the  necessity  of  a  mineral  base,  and  the  optimum 
amount  of  moisture  which  was  best  supplied  from  the  drainage  of 
stables. 

Early  Theories.— Even  though  the  process  had  reached  such  a 
high  state  of  development,  the  underlying  principles  were  entirely 
unknown  until  the  last  third  of  the  nineteenth  century.  At  this 
time  attempts  were  made  to  explain  the  oxidation  of  ammonia  to 
nitric  acid,  on  the  strength  of  certain  chemical  reactions  which 
could  be  brought  about  in  the  laboratory.  These  were  the  experi- 
ments of  Kuhlmann  and  Dumas.  The  first  investigator  found, 
on  passing  ammonia  and  air  through  a  heated  tube  containing 
a  platinum  sponge,  that  they  combined  with  the  formation  of 
ammonium  nitrate,  while  Dumas  found  that  nitric  acid  was  pro- 
duced when  air  and  ammonia  were  heated  to  100°  C.  with  moist- 
ened lime.  It  was  considered  possible  that  the  porosity  of  the 
soil  could  act  as  did  the  platinum  sponge  or  the  lime  of  the  soil 
act  in  a  manner  similar  to  that  used  in  Dumas'  experiments.  After 
the  discovery  of  ozone  by  Schonbein  this  substance  was  used  to 
explain  all  natural  processes  of  oxidation,  and  hence  naturally  the 
case  with  nitrification.  Mulder  stated  that  investigations  had 
shown  that  ozone  is  capable  of  oxidizing  ammonia  to  nitric  acid 
and  water,  and  that  it  is  probable  that  the  same  reaction  could 
take  place  in  the  soil,  the  soil  acting  merely  as  a  catalyzer  in  the 
reaction. 


THE  DAWN  OF  THE  BIOLOGICAL   THEORY  209 

It  may  be  seen  that  in  all  the  early  theories  it  was  supposed 
to  be  a  purely  chemical  process;  it  was  not  until  the  time  of  Pasteur 
that  the  biological  explanation  for  the  formation  of  nitrates  received 
any  support. 

It  is  interesting,  however,  to  note  the  careful  work  of  Bous- 
singault  in  the  years  between  1860  and  1878,  on  the  natural  occur- 
ring saltpeter  beds,  especially  those  of  Peru  and  Ecuador.  He 
raised  the  question:  "Have  not  the  nitrates  in  these  natural  deposits 
resulted  from  the  breaking  down  of  organic  substances  rich  in 
nitrogen?"  for  it  had  long  been  the  practice  to  use  blood,  urine, 
and  other  animal  refuse  for  the  production  of  nitrates.  For  this 
reason,  Boussingault  did  not  think  it  likely  that  the  gaseous  nitro- 
gen of  the  air  played  a  very  great  part  in  the  process  of  nitrifica- 
tion. In  order  to  test  this,  he  placed  soil  with  known  nitrogen 
content  in  100-liter  jars  and  allowed  them  to  remain  for  eleven 
years.  At  the  end  of  this  time  they  were  analyzed,  and,  in  spite 
of  the  fact  that  a  very  active  nitrification  had  taken  place,  there 
was  no  increase  in  the  total  nitrogen  of  the  soil.  From  this  he 
concluded  that  free  nitrogen  takes  no  part  in  the  formation  of 
nitrates,  but  that  they  result  from  the  organic  matter  of  the  soil. 
In  another  set  of  experiments  he  added  organic  manure  to  soil, 
sand,  and  chalk,  and  left  them  to  nitrify.  He  obtained  an  active 
nitrification  in  the  soil,  but  none  in  the  sand  or  chalk.  This  fact 
had  a  great  influence  on  the  old  theories  of  nitrification.  Why  this 
difference  if  the  soil  acts  merely  as  a  catalyzer?  It  may  be  said 
that  this  w:as  the  beginning  of  the  end  of  the  old  chemical  theories 
of  nitrification. 

The  Dawn  of  the  Biological  Theory.— At  this  time  (1878)  the 
work  of  Pasteur  was  beginning  to  take  firm  root.  There  had 
appeared  a  series  of  reports  on  fermentation,  one  of  the  earliest 
(1862)  being  on  the  fermentation  of  acetic  acid.  This  was  similar 
to  nitrification,  for  it  was  known  that  alcohol  could  be  oxidized 
to  acetic  acid  by  use  of  the  platinum  sponge.  In  fact  in  this  early 
publication  Pasteur  suggested  that  nitrification  was  due  to  a  fer- 
ment, and  soon  after  Mliller  observed  that  the  ammonia  in  sewerage 
is  rapidly  changed  into  nitrates,  but  no  corresponding  change  takes 
place  in  pure  ammonia  solution.  He  suggested  that  the  sewage 
probably  contained  a  ferment  which  was  absent  from  the  pure 
solution  prepared  in  the  laboratory.  He,  however,  took  no  steps 
to  prove  that  the  process  was  a  true  fermentation.  Between 
1871-75  Gilbert  noted  that  the  drainage  waters  from  the  Rothamsted 
experiment  fields  contained  more  nitrates  as  the  ammonium  salts 
applied  to  the  soil  increased. 

To  the  chemists,  Schlosing  and  Miintz,  belongs  the  credit  of 
establishing  by  experiment  the  fact  that  nitrification  is  a  biological 
process.     They  were  trying  to  ascertain  if  the  presence  of  humus  is 
14 


210  NITRIFICATION 

essential  in  the  purification  of  sewage  by  soil.  They  filled  a  glass 
tube  one  meter  long  with  ignited  quartz  sand  and  powdered  lime- 
stone. Sewage  passed  through  his  filter  at  first  unchanged,  but 
later  nitrates  began  to  appear,  and  soon  the  filtrate  contained 
no  ammonium  salts.  They  suspected  microorganisms  as  being 
the  active  agent,  and  hence  treated  the  contents  with  chloroform 
vapor.  Nitrification  entirely  ceased  and  was  not  renewed  for 
seven  weeks,  although  the  supply  of  chloroform  w^as  suspended. 
A  water  extract  of  fresh  garden  soil  added  to  the  tube  soon  restarted 
the  process. 

These  experiments  were  immediately  repeated  by  Warington  who 
confirmed  the  results  of  Schlosing  and  Miintz  and  showed  that: 
(1)  The  power  of  nitrification  could  be  communicated  to  media 
which  did  not  nitrify  by  simply  seeding  them  with  a  nitrifying 
substance;  (2)  the  process  of  nitrification  in  garden  soil  is  entirely 
suspended  by  the  presence  of  the  vapor  of  chloroform  or  carbon 
disulphid.  Since  these  early  experiments  much  additional  proof 
has  been  furnished  by  investigators  showing  that  the  process  of 
nitrification  both  in  soils  and  waters  is  undoubtedly  due  to  living 
microorganisms.  Some  of  these  are  the  limits  of  temperature 
within  which  nitrification  is  possible,  the  necessity  of  a  suitable 
food,  and  finally  the  isolation  of  specific  organisms  having  the 
power  of  producing  nitrates. 

Schlosing  and  Miintz  were  unable  to  isolate  any  specific  ferment 
capable  of  causing  nitrification,  but  the  true  nature  of  the  process 
not  being  known,  many  investigators  turned  to  this  phase  of  the 
work  and  the  race  to  see  who  would  first  reach  the  coveted  goal  be- 
came intensely  interesting.  Celli-Zuco  and  Heraeus,  in  1886,  suc- 
ceeded in  isolating  from  water  rich  in  nitrates  a  number  of  forms  of 
bacteria  which  they  considered  possessed  very  feeble  nitrifying 
properties.  Inasmuch  as  the  nitric  acid  in  their  cultures  may  have 
been  absorbed  from  the  air,  and  as  they  did  not  succeed  in  isolat- 
ing and  proving  any  organism  to  be  capable  of  nitrification  their 
experiments  were  considered  to  be  inconclusive.  Frank  attempted 
a  similar  isolation  but  without  success,  and  he  even  concluded 
that  nitrification  was  not  due  to  the  direct  action  of  microorganisms 
but  was  a  purely  chemical  process.  But  this  view  was  opposed 
by  a  number  of  writers,  notably  Landolt,  Platt,  and  Baumann. 

Warington  and  Frankland  studied  a  large  number  of  soil  organ- 
isms, but  neither  was  able  to  find  any  which  produced  active 
nitrification.  Frankland  continued  to  maintain  that  the  nitrify- 
ing organism  was  present  in  soil,  and  in  1890  succeeded  in  isolating 
a  spherical  organism  about  0.8  M  in  diameter  which  possessed  the 
power  of  converting  ammonium  salts  into  nitrites,  but  not  into 
nitrates.  The  separation  was  made  by  means  of  the  dilution 
method,  using  only  inorganic  salts. 


ISOLATION  OF  NITRIFYING  FERMENTS  211 

Isolation  of  Nitrifying  Ferments.— The  only  definite  result  which 
had  been  reached  up  to  1890  was  that  there  must  exist  in  soil 
microorganisms  which  possess  the  power  of  nitrifying.  However, 
up  to  the  time  Winogradsky  took  up  the  subject  all  attempts  to 
isolate  such  organisms  had  proved  futile.  His  previous  experience 
had  been  such  as  to  confirm  his  belief  in  such  organisms.  He  had 
discovered  microorganisms  which  oxidized  hydrogen  sulphid 
(sulpho-bacteria)  and  an  iron  compound  (ferro-bacteria).  He 
reasoned  that  it  was  extremely  probable  that  organisms  should  exist 
in  water  and  in  soil  capable  of  availing  themselves  of  the  abundant 
energy  which  would  come  from  the  oxidation  of  the  ammonium 
compounds  contained  therein.  He  considered  that  the  number  of 
such  species  would  be  small  and  that  the  way  to  secure  and  study 
such  organisms  would  be:  (1)  To  find  a  medium  and  condition 
under  which  they  would  thrive  and  by  which  the  growth  of  deni- 
trifying organisms  would  be  discouraged;  (2)  to  continue  the  culti- 
vation by  such  a  method  long  enough  to  eliminate  for  the  most 
part  other  organisms;  and  (3)  when  the  cultures  of  the  oxidizing 
organisms  should  have  been  obtained  reasonably  pure  and  their 
nitrification  of  ammonia  active,  to  proceed  to  isolate  the  various 
organisms  and  study  the  character,  and  especially  the  nitrifying 
power  of  each  in  pure  cultures.  He  knew  that  all  previous  attempts 
to  isolate  the  organism  on  gelatin  plates  had  failed  and  he  con- 
sidered that  probably  the  organism  would  not  grow  on  gelatin. 
He  began  by  working  with  two  soils,  one  rich  in  organic  matter, 
the  other  poor,  but  both  rich  in  carbonate,  and  soon  he  learned  that 
organic  matter  hindered  nitrification.  After  considerable  work  he 
finally  adopted  a  medium  having  the  following  composition: 

Water  of  Lake  Zurich "...  1000  c.c. 

Ammonium  sulphate 1  gm. 

Potassium  phosphate 1  gm. 

Magnesium  carbonate 0.5-1  gm.  per  100  c.c. 

When  such  a  medium  was  inoculated  with  suitable  material, 
nitrification  was  so  active  that  after  fifteen  days  every  trace  of 
ammonia  had  disappeared,  whereas  check  solution  contained  only 
slight  traces  of  nitric  acid. 

Cultures  were  continued  and  repeated  in  this  medium  for  three 
months,  when  he  considered  that  only  the  organisms  suited  to 
this  medium  had  survived.  These  were  inoculated  on  to  gelatin 
plates  and  five  species  of  organisms  were  found  to  form  colonies— 
three  bacteria,  one  oidium,  and  the  fifth  a  peculiar  organism  which 
he  designated  as  "fungus."  None,  however,  possessed  the  power 
of  converting  ammonia  into  nitric  acid. 

In  the  liquid  cultures  it  was  observed  that  a  very  thin  film  gradu- 
ally formed  on  the  surface  of  the  culture  and  at  times  a  slight 


212  NITRIFICATION 

cloudiness  of  the  solution  was  noted.  The  latter  disappeared 
after  a  time  and  a  microscopic  examination  showed  it  to  be  due 
to  the  presence  of  an  oval  somewhat  spindle-shaped  organism 
which  moved  about  very  rapidly.  Nitrification  was  at  the  same 
time  very  active.  It  was  thought  that  the  film  on  the  surface 
might  contain  the  nitrifying  organisms  as  the  acetic  acid  bacteria 
and  other  oxidizing  ferments  work  at  the  surface  where  plenty  of 
oxygen  can  be  obtained,  but  tests  with  this  gave  negative  results. 
The  plan  of  work  was  then  somewhat  changed.  The  attempt 
was  made  to  cultivate  the  nitrifying  organisms  more  abundantly. 
To  this  end  a  quantity  of  ammonium  sulphate  was  added  to  the 


FIG.  30. — Surface  colonies  of  nitrosomonas  on  silicic  acid  gelatin,  stained  with 
Fuchsin  without  removal  from  the  gelatin.      X   1200.     (After  Gibbs:  Soil  Science.) 

nitrifying  cultures,  and  the  process  of  nitrification  thus  continued 
in  the  culture  for  some  time.  A  change  was  noticed  in  the  mag- 
nesium carbonate  at  the  bottom  of  the  solution  which  gradually 
assumed  a  grayish  color  and  a  gelatinous  consistency.  By  shaking 
the  solution  vigorously  this  mass  was  broken  up  into  small  flakes, 
which  a  microscopic  examination  showed  to  consist  of  transparent 
particles  of  the  salt  covered  with  a  mass  of  oval  bacteria,  identical 
in  form  with  those  which  had  previously  been  noticed  as  the  cause 
of  the  cloudiness.  These  bacteria  seemed  to  be  on  the  particles 
exclusively  and  not  on  the  walls  of  the  flask,  am}  slowly  enveloped 
the  salt  which  was  finally  dissolved. 


ISOLATION  OF  NITRIFYING  FERMENTS 


213 


A  culture  medium  was  then  prepared  which  was  free  from  every 
trace  of  organic  matter.  On  being  inoculated,  nitrification  took 
place  in  this  energetically,  and  it  was  found  to  contain  large  num- 


FIG.  31. — Nitrobacteria  from  cultures  in  liquid  medium  stained  with  carbol  Fuchsin. 
X  2500.     (After  Gibbs:  Soil  Science.) 

bers  of  the  oval  bacteria,  as  well  as  the  fungus  form  previously 
noticed,  but  the  other  forms  had  all  disappeared.  The  fungus 
remained  constant,  and  all  attempts  to  cultivate  it  out  were  unsuc- 
cessful. 


214 

The  research  was  thus  brought  to  the  stage  where  it  seemed 
probable  that  the  oval  bacteria  might  be  the  nitrifying  agents. 
To  test  their  nature  and  action  satisfactorily  the  removal  of  the 
sprouting  fungus  was  called  for.  To  accomplish  this,  Winogradsky 
resorted  to  a  very  ingenious  though  a  simple  device.  The  fungus 
would  develop  in  gelatin;  the  bacteria  would  not.  Small  particles 
of  the  carbonate,  more  or  less  enveloped  by  the  bacteria,  were 
taken  from  the  bottom  of  the  culture  flask  by  means  of  a  capillary 
tube  and  placed  in  a  large  flask  of  sterilized  water.  The  contents 
of  the  flask  were  then  well  shaken  and  a  gelatin  plate  inoculated 
with  drops  of  the  liquid,  the  particles  of  carbonate  serving  to  indi- 
cate the  places  where  the  gelatin  had  been  inoculated.  In  some 
of  these  the  fungus  developed.  Inoculations  of  the  culture  liquid 
from  the  other  spots  failed  to  yield  the  fungus  but  developed  the 
bacteria.  By  this  method  of  "inverse  gelatin  culture"  the  bacteria 
were  obtained  pure.  Liquid  cultures  inoculated  with  the  bacteria 
oxidized  ammonia  rapidly.  The  inference  was  that  the  bacteria 
were  the  nitrifying  organisms  of  the  soil. 

Winogradsky  describes  the  nitrite-forming  organisms  as  of  oval 
form,  about  1.1  to  1.8  /*  long  and  0.9  to  1  /x  wide,  usually  at  rest  but 
at  'times  capable  of  motion  and  dividing  perpendicularly  to  the 
longest  axis.  He  places  it  in  a  genus  by  itself,  which  he  calls 
Nitrosomonas. 

The  nitrate-forming  organism,  nitrobacter,  is  0.3  to  0.4  ju  wide 
and  about  1  ju  long.  The  cells  occur  singly  or  in  pairs  and  occa- 
sionally in  threes.  They  are  spindle-slaped,  non-motile,  and 
possess  a  capsule  which  makes  them  difficult  to  stain. 

By  way  of  comparing  the  activity  of  the  nitrobacter  with  that 
of  the  ferments  as  they  actually  occur  in  soil,  Winogradsky  made 
a  series  of  experiments  to  compare  the  amount  of  nitrification  in 
his  culture  liquid  with  that  observed  by  Schlosing  in  a  soil  to 
which,  however,  more  oxygen  had  access  than  was  the  case  with 
Winogradsky's  liquid. 

While  in  Schlosing's  experiments  by  the  use  of  200  grams  of 
earth,  3.4,  9,  and  4.1  nag.  of  nitrogen,  respectively,  were  nitrified, 
Winogradsky's  pure  cultures  of  bacteria  nitrified  860  mg.  of  ammo- 
nium sulphate  in  twenty-seven  days  and  930  mg.  in  thirty  days. 
Therefore,  during  the  period  at  which  nitrification  was  most  ener- 
getic there  would  be  formed  about  7.2  mg.  of  nitrogen  per  day. 
-\  Winogradsky  further  investigated  the  interesting  and  very 
remarkable  fact  previously  cited,  that  the  nitrobacter,  although 
containing  no  chlorophyll,  grows  and  is  able  to  multiply  in  a  solu- 
tion entirely  free  from  organic  matter. f  To  prove  this  fact  beyond 
doubt  he  prepared  a  culture  medium  absolutely  free  from  every 
trace  of  organic  matter  by  using  twice  distilled  and  tested  water 
and  salts  which  had  been  carefully  purified  by  recrystallization. 


ISOLATION  OF  NITRIFYING  FERMENTS  215 

He  thoroughly  removed  all  organic  matter  from  the  glass  dishes 
and  apparatus  to  be  used,  and  inoculated  separate  portions  ot 
the  medium  with  the  nitrobacter.  The  cultures  developed  nor- 
mally in  the  dark  as  well  as  in  the  light.  To  gain  an  idea  of  the 
extent  of  the  assimilation  of  carbon,  the  carbon  in  the  organic 
matter  which  had  been  formed  by  the  organism  in  its  growth  was 
determined  by  analysis.  Four  cultures  contained  10.2,  7.1,  4.6, 
and  4.8  mg.,  respectively,  of  assimilated  carbon,  and  in  these 
cultures  928,  604,  and  83.5  mg.,  respectively,  of  nitric  acid  had 
been  formed.  This  seemed  to  leave  no  doubt  that  nitrobacter  is 
able  to  assimilate  the  carbon  of  carbonic  acid. 

Later,  in  1891,  Warington,  in  a  solution  containing  mineral 
salts,  obtained  after  repeated  generation  a  culture  which  nitrified 
vigorously.  This  contained  no  organisms  which  would  grow  on 
gelatin  and  was  regarded  by  him  as  containing  only  nitrifying 
bacteria.  The  organisms  thus  obtained  were  oval  in  form  and 
seldom  1  micromillimeter  thick  and  only  slightly  longer. 

At  this  time  Winogradsky  made  a  decided  improvement  in  the 
separation  of  the  nitrifying  organism  from  solutions  by  use  of  the 
Kuhne  gelatin  silica  medium.  The  nutrient  basis  of  this  medium 
as  used  by  Winogradsky  was  composed  of  ammonium  sulphate,  0.41 
grams;  magnesium  sulphate,  0.05  grams;  potassium  phosphate,  0.1 
gram;  sodium  carbonate,  0.6-0.9  gram;  calcium  chlorid,  a  trace; 
and  water,  100  c.c. 

The  inoculation  of  the  plates  took  place  either  by  mixing  the 
inoculating  material  with  the  above  solution  before  the  addition 
of  gelatinous  silica,  or  it  was  made  as  a  streak  or  smear  culture 
on  the  already  hardened  material.  In  this  way  the  nitrifying 
organisms  developed  distinct  colonies  from  which  pure  cultures 
were  obtained. 

The  investigations  of  Winogradsky  and  simultaneously  of  War- 
ington showed  the  following:  (1)  That  in  the  soil  the  nitrifying 
process  was  effected  by  two  distinct  but  closely  related  organisms, 
the  one  converting  ammonia  into  nitrous  acid  and  nitrite  and  the 
other  changing  the  nitrites  into  nitrates.  (2)  That  these  two 
processes  follow  one  another  in  such  rapid  succession  that  the 
production  of  nitrites  is  only  a  transitory  phenomenon,  so  that 
if  both  the  nitrite  and  nitrate  organism  be  added  to  sterilized  soil 
the  process  is  completed  in  the  natural  way,  only  the  merest  traces 
of  nitrous  acid  appearing. 

If  to  a  mineral  solution  containing  ammonium  salts,  a  pure  cul- 
ture of  nitrosomonas  be  added,  only  nitrites  will  appear  and  these 
will  remain  unchanged  in  the  absence  of  the  nitrobacter.  If, 
however,  the  two  organisms  be  added  simultaneously  nitrates  will 
be  rapidly  formed. 


216  NITRIFICATION 

According  to  Kaserer  there  is  an  organism  which  can  oxidize 
ammonia  direct  to  nitric  acid,  but  so  far  this  has  not  been  confirmed. 

In  1892  Winogradsky  studied  the  nitrifying  organisms  of  the 
soil  from  a  number  of  different  localities.  Those  from  several 
parts  of  Europe,  from  Africa,  and  from  Japan,  which  he  considers 
to  be  the  same  organism,  he  names  Nitrosomonas  europea.  A  second 
form  from  Java  soil  differing  from  the  first,  he  names  Nitrosomonas 
javenensis.  Both  of  these  comprise  the  nitrate  ferments  of  Wino- 
gradsky, the  second  nitrate  ferment  was  isolated  by  Winogradsky 
from  Quito  soil  and  differs  from  the  first  not  only  as  to  size,  as 
above  mentioned,  but  also  by  entirely  lacking  the  motility  common 
to  the  latter. 

In  1895  Burri  and  Stutzer  isolated  from  soil  a  nitrate  organism 
with  properties  akin  to  the  Quito  bacillus  of  Winogradsky.  It  was 
a  motile  organism,  0.75  -  -  1.5  x  0.5  micromillimeters,  growing 
on  gelatin  which  it  liquefied,  said  organism,  according  to  these 
workers,  being  able  to  convert  nitrites  into  nitrates,  but  losing 
such  power  when  grown  on  organic  media. 

The  results  of  Burri  and  Stutzer,  so  contrary  to  those  of  Wino- 
gradsky, brought  forth  a  vigorous  rejoinder  from  the  latter.  In 
this  Winogradsky  stated  that  he  tested  the  same  earth  used  by 
Burri  and  Stutzer  and  isolated  therefrom  his  own  nitrosomonas,  and 
that  the  latter  when  tested  in  bouillon,  meat  peptone,  gelatin,  and 
agar  failed  to  grow.  He,  therefore,  regards  the  German  work  as 
erroneous. 

In  1897  Stutzer  and  Hartleb  appeared  with  a  still  more  startling 
series  of  discoveries  in  which  they  not  only  maintained  the  ability 
of  the  nitrifying  organisms  to  grow  in  organic  media,  but  also 
showed  that  the  latter  possessed  a  polymorphic  habit  never  imag- 
ined in  this  or  any  other  like  group  in  the  whole  domain  of  mycol- 
ogy—the ability  of  simple  coccoid  or  rod-shaped  forms  to  develop 
into  filaments  or  even  into  branched  forms,  with  the  further  pro- 
duction of  true  gonidia  and  other  even  more  highly  organized 
fructification  bodies. 

Gartner  discussed  the  work  of  Burri,  Stutzer  and  Hartleb  on  the 
polymorphism  of  the  nitrifying  organism,  and  from  presumably 
pure  cultures  of  the  latter's  nitrifying  ferment  was  able  to  isolate 
thirteen  different  microorganisms,  including  a  fungus  form  (Schim- 
melpilz),  thus  proving  their  impure  character.  Furthermore, 
Gartner  showed  that  these  several  organisms,  when  once  separated 
in  their  pure  state,  retained  their  fixed  character,  with  no  tendency 
to  polymorphism,  and  indicated  none  of  those  transition  stages 
from  bacteria  to«  fungi  noted  by  Stutzer.  Again,  none  of  these 
isolated  organisms  possessed  the  power  to  convert  ammonia  into 
nitrites.  C.  Fraenkel  simultaneously  isolated  from  Burri  and 
Stutzer's  cultures  11  different  organisms,  including  7  bacilli 


DISTRIBUTION  217 

2  streptothrices,  and  2  fungi  (a  Fadenpilz  and  a  Schimmelpilz) . 
These  showed  no  polymorphism,  but  all  retained  constant  char- 
acters. 

In  1902  Chester  summarized  the  knowledge  on  nitrification  as 
follows: 

1.  That  nitrification  in  the  soil  is  caused  by  a  distinct  or  rather 
by  two  distinct  organisms  possessing  certain  definite  characters. 

2.  That  these  organisms  will  not  grow  in  the  presence  of  any 
considerable   amount   of   organic   matter,   and   that  all  reported 
attempts  to  cultivate  them  on  ordinary  organic  media  are  without 
authentication. 

3.  That  the  above  nitrifying  organisms  are  found  abundantly 
in  all  cultivated  soils  and  in  ordinary  soil  water  containing  a  due 
proportion  of  ammonium  carbonate,  sulphate,  etc.,  they  find  a  favor- 
able medium  for  their  development. 

4.  That  the  result  of  such  development  is:    (a)  The  conversion 
of  ammonia  into  nitrous  acids  through  the  agency  of  the  nitrous 
organism;  and  (b)  the  immediate  conversion  of  the  previous  nitrous 
into  nitric  acid  by  means  of  the  equally  abundant  nitric  ferment. 

Distribution.— Probably  the  nitrifying  bacteria  were  some  of  the 
first  living  organisms  to  appear  upon  this  planet,  and  even  yet 
they  act  as  the  pioneers  preparing  the  soil  for  other  plants.  Muntz 
has  found  the  decayed  rocks  of  Alpine  summits,  where  no  other 
life  exists,  swarming  with  the  nitrifying  ferments.  The  limestones 
and  micaceous  schists  of  the  Pic  du  Midi,  in  the  Pyrenees,  and  the 
decayed  calcareous  schists  of  the  Faulhorn,  in  the  Bernese  Ober- 
land,  offer  good  examples  of  this  kind.  The  organisms  draw  their 
nourishment  from  the  nitrogen  compounds  brought  down  in  snow 
and  rain;  they  convert  the  ammonia  into  nitric  acid,  and  this  in 
turn  corrodes  the  calcareous  portions  of  the  rock.  Stiitzer  and 
Hartleb  have  observed  a  similar  decomposition  of  cement  by 
nitrifying  bacteria. 

The  nitrifying  bacteria  appear  to  be  very  widely  distributed 
Muntz  and  Aubin  have  observed  their  presence  not  only  in  all 
cultivated  soils  which  they  have  examined,  but  also  in  those  of 
deserts.  They  are  not  usually  found  in  the  air  or  in  rain  water. 
River  water  and  sewage  contain  them.  They  are  usually  present 
in  well  waters.  In  the  case  of  deep  wells  their  origin  is  due  to 
surface  soil  or  to  drainage  from  the  surface  soil  which  has  found 
its  way  into  the  well,  the  water  of  deep  wells  not  being  their  natural 
habitat.  Thomasen  found  the  nitrite  organism  in  samples  of 
ooze  from  the  bottom  of  the  Kiel  Fjord,  but  not  in  the  sea  water 
nor  on  the  Plankton  or  the  fixed  algae.  It  was  also  found  in  similar 
samples  of  soil  from  the  vicinity  of  Helgoland  and  in  slime  from 
the  bottom  of  the  Bay  of  Naples,  but  only  in  samples  taken  near 
the  land. 


218  NITRIFICATION 

Warington  failed  to  find  the  nitrifying  ferments  in  a  clay  soil 
below  eighteen  inches,  and  this  is  in  keeping  with  the  findings  of 
Ladd  at  North  Dakota.  For  a  long  time  it  was  considered  that 
they  are  found  only  in  the  surface  soil,  but  in  1906  Welbel  pre- 
sented results  with  soils  where  nitrification  is  almost  as  active  in 
the  subsoil  as  in  the  surface  soil  when  the  subsoil  is  aerated.  In 
1912  C.  B.  Lipman  found  them  often  to  a  depth  of  five  or  six  feet 
in  soils  of  the  arid  regions.  In  one  case  soil  from  the  eight-foot 
depth  showed  a  vigorous  nitrifying  power.  The  author  found  soil 
from  second  and  third  foot-sections  to  nitrify  dried  blood  quite 
readily,  as  is  shown  below: 

Irrigated  Dry-farm 

Depth  Soil  Soil 

First  twelve  inches 19.39  5.25 

Second  twelve  inches        ........        2.70  2.41 

Third  twelve  inches 1.98  1 . 55 

These  are  the  averages  of  several  hundred  examinations,  and 
many  soils  which  were  fairly  heavy  clays  showed  active  nitrifica- 
tion in  the  second  and  third  foot-sections.  This  great  difference 
observed  in  the  arid  regions  is  due  mainly  to  a  better  aeration  of 
these  subsoils  which,  because  of  the  peculiar  climatic  conditions, 
the  arid  soils  are  not  as  rich  in  clay  as  are  the  subsoils  of  the  humid 
regions.  Moreover,  the  plants  in  the  arid  regions  root  to  a  great 
depth  in  search  of  water.  These  decaying  roots  loosen  up  the 
subsoil  and  also  furnish  food  for  bacterial  growth. 

Reaction  of  Media.— Boussingault  long  ago  observed  that  many 
forest  soils  do  not  contain  nitrates,  and  later  this  was  verified  by 
Breal  and  others.  We  now  know  that  the  absence  of  nitrates  is 
due  to  the  acid  reaction  of  a  soil  which  contains  an  excess  of  organic 
matter.  The  nitric  ferment  does  not  act  in  an  acid  medium;  hence, 
we  have  the  explanation  of  the  great  benefit  derived  from  the  use 
of  basic  substance. 

Experiments  by  Wiley  and  Elwell  in  which  solutions  containing 
calcium  chlorid  and  water  were  seeded  with  nitrifying  ferments 
continued  to  nitrify  until  the  medium  contained  an  acidity  equiva- 
lent to  4  c.c.  of  normal  acid  per  100. 

Dumont  and  Crochetelle's  experiments  are  of  the  same  order. 
They  took  soil  which  had  been  in  grass  from  time  immemorial  and 
which  contained  6.84  per  cent,  of  humus.  This  was  treated  with 
variable  quantities  of  potassium  carbonate.  It  was  stirred  and 
watered  several  times  during  the  experiment  and  after  one  month 
the  nitrates  were  extracted  with  the  following  results:  nitric  nitro- 
gen, per  1000  grams,  of  soil  without  addition  of  potassium  car- 
bonate, 70  mgs.;  with  1  gram  of  potassium  carbonate,  160  rags.; 
with  2  grams  of  potassium  carbonate,  230  mgs. ;  with  3  grams,  250 
mgs.;  with  4  grams,  130  mgs.;  with  5  grams,  73  mgs.  In  similar 


REACTION  OF  MEDIA  219 

experiments,  Kochenavski  demonstrated  that  potassium  carbo- 
nate is  more  efficient  in  this  regard  than  is  calcium  carbonate, 
probably  because  the  potassium  acts  as  a  food  in  addition  to  the 
neutralizing  of  the  acid.  ,Owen  has  found  magnesium  carbonate 
even  more  efficient  than  potassium  carbonate,  and  this  is  in  keep- 
ing with  the  findings  of  Lyon,  and  Bizzell  and  White.  Pangan- 
iban's  findings  appear  to  differ  from  these,  for  he  claims  that  liming 
greatly  increases  nitrification  only  when  the  limestone  contains  little 
magnesium  carbonate.  The  soil  of  the  Utah  Greenville  farms  con- 
tains 16.88  per  cent,  of  lime  (CaO)  and  6.1  per  cent,  magnesium 
(MgO),  and  they  nitrify  ammonium  sulphate,  dried  blood,  and 
cottonseed  meal  readily. 

The  carbonates  are  not  the  only  substances  in  the  soil  which 
serve  as  bases  for  nitrification,  since,  according  to  Ashby,  a  marked 
nitrification  of  ammonium  salt  can  be  brought  about  in  the  presence 
of  ferric  hydrate,  either  in  the  freshly  precipitated  state  or  as 
"iron  rust."  In  solutions,  however,  nitrification  is  not  completed 
where  iron  is  the  only  base,  probably  because  the  ferric  nitrite  or 
nitrate  formed  dissociates  and  the  solution  becomes  acid. 

The  double  ammonium  combination  formed  by  the  absorption 
of  ammonium  salts  by  modelling  clay  can  most  probably  be  nitri- 
fied in  the  absence  of  any  base,  but  the  corresponding  combination 
with  peat  undergoes  no  nitrification  in  the  absence  of  a  base. 

One  of  the  functions  of  the  base  in  nitrification  is  to  form  ammo- 
nium carbonate,  and  the  facility  with  which  nitrification  is  set  up 
by  different  carbonates  depends  upon  the  rapidity  with  which 
they  can  react  with  a  neutral  ammonium  salt  to  produce  ammo- 
nium carbonate.  This  reaction  is  greater  with  magnesium  car- 
bonate than  with  calcium  carbonate,  but  is  almost  absent  with 
copper  carbonate. 

The  quantity  of  lime  which  must  be  added  to  a  soil  for  maximum 
nitrification  varies  with  the  original  reaction  of  the  soil  and  the 
fertilizer  to  be  nitrified;  ammonium  sulphate  requires  more  than 
bone  meal,  cottonseed  meal,  or  dried  blood. 

There  should  always  be  an  excess  of  the  base  present,  for  Fischer 
found  that  the  theoretical  amount  of  lime  (200  grams  of  calcium 
carbonate)  required  for  the  nitrification  of  ammonium  sulphate 
(132.7  grams)  was  not  sufficient  for  complete  nitrification,  but 
about  three  and  one-half  times  the  theoretical  amount  was  required. 
Even  much  larger  quantities  of  either  magnesium  carbonate  or 
calcium  carbonate  may  be  used  without  ill  effect,  but  large  quan- 
tities of  quicklime  may  cause  a  rapid  burning  out  of  the  organic 
matter  and  even  volatilization  of  ammonia  and  may  even  stop 
nitrification.  For,  while  nitrification  takes  place  in  a  feebly  alka- 
line medium,  yet  the  presence  of  anything  beyond  a  small  quan- 
tity of  an  alkaline  salt  is  a  hindrance  to  the  process,  and  a  large 


220  NITRIFICATION 

amount  will  check  it  entirely.  Thus  Warington  found  that  the 
presence  of  0.032  per  cent,  of  bicarbonate  of  soda  distinctly  retarded 
nitrification,  and  in  the  presence  of  0.096  per  cent,  nitrification  was 
only  barely  possible.  The  same  author  also  showed  that  the 
presence  of  0.0477  per  cent,  of  ammonia  in  urine  rendered  it  unnitri- 
fiable.  Dumont  and  Crochetelle  found  that  potassium  carbonate 
added  to  soil  at  the  rate  of  from  1  to  2.5  grams  per  1000  grams  of 
soil  markedly  increased  nitrification,  but  larger  applications  of  the 
salt  progressively  diminished  the  rate  of  nitrification,  and  that  the 
addition  of  8  grams  per  1000  grams  of  soil  completely  checked  it. 
A  heavy  dose  of  lime  by  unduly  increasing  the  alkalinity  of  the 
soil  may  at  first  check  or  suspend  nitrification  until  the  said  lime 
has  been  converted  into  carbonate.  This,  however,  takes  place, 
rapidly,  diminishing  in  turn  its  strong  alkaline  properties  and  per- 
mitting nitrification  to  commence  more  actively  than  before. 

Food  Requirements  of  Nitrifiers.— The  nitrifying  organisms  require 
the  same  elements  as  do  other  bacteria,  and  hence  will  be  considered 
in  this  chapter  only  in  a  very  general  way,  except  in  regard  to 
the  source  of  the  required  elements. 

Winogradsky  found  that  the  nitrosomonas  were  able  to  grow  in 
a  medium  consisting  of  2.25  grams  of  ammonium  sulphate,  2  grams 
of  common  salt,  and  1  grams  of  magnesium  carbonate  in  1  liter  of 
well  water.  For  the  nitrobacter  the  ammonia  is  replaced  by 
sodium  nitrite.  In  media  such  as  the  above,  devoid  of  organic 
carbon,  the  nitrifying  organisms  are  able  to  function  in  the  dark 
and  form  from  the  inorganic  carbon,  organic  carbon  compounds. 
He  proved  by  numerous  quantitative  determinations  that  during 
nitrification  an  increase  in  the  amount  of  carbon  compounds  tafyes 
place.  "Since  this  bound  carbon  in  the  cultures  can  have  no 
other  source  than  the  carbon  dioxid  and  since  the  process  itself 
can  have  no  other  cause  than  the  activity  of  the  nitrifying  organ- 
ism, no  other  alternative  was  left  but  to  ascribe  to  it  the  power  of 
assimilating  carbon  dioxid. 

"Since  the  oxidation  of  ammonia  is  the  only  source  of  chemical 
energy  which  the  nitrifying  organisms  can  use,  it  is  a  priori  that 
the  yield  in  assimilation  must  correspond  to  the  quantity  of  oxi- 
dized nitrogen.  It  turned  out  that  an  approximately  constant 
ratio  exists  between  the  values  of  assimilated  carbon  and  those  of 
oxidized  nitrogen."  This  is  illustrated  by  the  following  results: 

No.  5.  No.  6.  No.  7.  No.  8. 

Oxidized  nitrogen        .722.0  506.1  .        928.3  815.4 

Assimilated  carbon      .        19.7  15.2  26.4  22.4 

Ratio — nitrogen:  carbon   36.6  33.3  35.2  36.4 

It  is  evident  that  1  part  of  assimilated  carbon  corresponds  to 
about  35.4  parts  of  oxidized  nitrogen  or  96  parts  of  nitrous  acid. 


FOOD  REQUIREMENTS  OF  NITR1FIERS  221 

More  recently,  Coleman  using  pure  cultures  of  nitrate  producers 
obtained  ratios  varying  from  40  to  44. 

Now  there  are  two  sources  of  carbon  dioxid  which  are  available 
to  the  nitrifying  organisms— one,  the  carbonate,  which  is  present 
in  the  soil;  the  other,  the  carbon,  in  the  air.  According  to  Wino- 
gradsky,  the  carbonate  supplies  the  carbon  for  the  bacterial  growth, 
it  being  liberated  by  means  of  the  acids,  which  they  produce.  On 
the  other  hand,  Godlewski  considered  that  it  is  chiefly  from  the 
atmosphere  that  the  carbon  dioxid  requisite  for  the  construction  of 
new  cellular  substance  is  derived.  He  found  that  development 
did  not  occur  in  cultures  containing  magnesium  carbonate  when 
air  free  from  carbon  dioxid  was  admitted,  and  concluded  that: 
(1)  Nitrosomonas  placed  in  a  pure  mineral  solution  are  unable  to 
assimilate  the  carbon  of  magnesium  carbonate;  (2)  it  is  very  improb- 
able that  the  nitrobacter  derive  their  carbon  from  the  organic 
substances  of  the  air;  (3)  it  is  very  probable  that  these  organisms 
find  the  carbon  which  they  need  in  the  free  carbonic  acid  or  in  the 
carbonic  acid  of  bicarbonates.  But  Owen,  after  careful  experi- 
ments in  which  he  used  a  specially  devised  flask  for  the  elimination 
of  the  carbon  dioxid  of  the  air,  concluded  that  "the  nitrifying 
organisms  of  the  soil  do  not  depend  to  any  appreciable  extent  on 
the  carbon  dioxid  of  the  air  for  their  carbon  supply."  Hence,  the 
evidence  seems  to  be  that  the  organisms  under  appropriate  condi- 
tions possess  the  power  of  utilizing  either  source  of  carbon. 

The  nitrite  bacteria  obtain  their  nitrogen  both  for  oxidation  in 
the  production  of  energy  and  as  building  material  from  ammonia 
preferably  in  the  form  of  ammonium  carbonate.  They  are,  how- 
ever, according  to  Ashby,  able  to  utilize  the  double  ammonium 
combination  formed  through  the  absorption  of  ammonium  salts 
by  modelling  clay,  but  the  corresponding  combination  with  peat 
undergoes  no  nitrification  in  the  absence  of  a  base.  However, 
according  to  Marcille,  the  nitrogen  of  ammonium  phosphate  is  not 
so  readily  transformed  into  nitrous  acid  as  is  that  of  ammonium 
sulphate.  Yet  the  phosphate  appears  to  furnish  a  much  more  favor- 
able medium  for  the  transformation  of  nitrites  into  nitrates  than 
does  the  sulphate. 

While  calcium  cyanamid  is  nitrified  when  added  to  a  soil,  it  is 
not  until  it  has  been  transformed  into  ammonia  by  other  bacteria, 
chief  among  which  are,  according  to  Lohnis,  B.  putidum,  B. 
mycoides,  B.  vulgare  var.,  B.  zopfii  lepsiense  (n.sp.),  B.  kirchneri 
(n.  sp.),  B.  megatherium,  B.fluorescens,  B.  subtilis,  B.  ellenbachensis 
arid  B.  vulgare.  According  to  Boullanger,  the  nitrous  organism 
does  not  attack  hydroxylamin  hydrochlorid. 

Excessive  quantities  of  ammonia  or  ammonium  salts  hinder  the 
multiplication  |  of |  nitrifying  organisms  but  do  not  interfere  with 
the  action  of  those  already  present.  Boullanger  and  Massol  found 


222  NITRIFICATION 

the  minimum  retarding  amount  of  ammonia  to  be  about  2  parts 
per  million.  It  is  seldom  sufficient  ammonia  accumulates  in  soils 
under  natural  conditions  to  interfere  with  the  multiplication  of 
nitrifying  bacteria. 

Just  as  all  organic  nitrogen  must  be  ammonified  before  it  can 
be  changed  by  the  nitrosomonas  to  nitrous  acid,  so  all  ammonia 
compounds  vmust  be  oxidized  to  nitrous  acid  before  the  nitrobacter 
can  convert  them  into  nitric  acid.  The  nitrite  organism  readily 
oxidizes  nearly  all  nitrites  in  solutions  containing  0.5  to  1  gram 
per  liter,  but  larger  quantities  of  the  nitrites  are  toxic  even  to  the 
nitromonas. 

Organic  Matter.  —  Winogradsky  early  learned  that  the  nitrifying 
organisms  will  not  grow  in  a  medium  containing  soluble  organic 
matter,  and  since  that  time  numerous  experiments  have  been 
made  to  account  for  this  apparent  discrepancy.  It  was  well  known 
that  nitrification  takes  place  in  the  soil  and  compost  which  contains 
organic  material.  Hence,  the  theory  was  soon  advanced  that 
organic  matter  in  the  form  of  humus  is  not  injurious  and  may 
actually  be  beneficial,  as  is  illustrated  by  the  work  of  Smirnov: 

Humus  NITRIC  NITROGEN  IN  100  GRAMS  SOIL 

Per  cent.  At  beginning         After  19  Days         After  36  Days         After  73  Days 

0.42       .      .      .      .      1.5mg.  14.0mg.  25.5mg.  28.0  ing. 

3.55       ....      0.5  21.0  38.0  53.0 

Miintz  later  concluded  that  humus  even  in  larger  quantities 
does  not  interfere  with  nitrification,  but  on  the  other  hand  it  is 
favorable  to  it.  Nor  is  an  abundance  of  humus  a  necessary  condi- 
tion to  nitrification,  since  soils  poor  in  this  constituent  gradually 
develop  intensive  nitrification.  He  considers  that  the  humus 
favors  the  multiplication  of  the  nitrifying  organisms  and  a  soil 
which  contains  a  large  amount  of  humus  is  more  abundantly  sup- 
plied with  these  organisms  and  more  apt  to  enter  into  rapid  nitri- 
fication. 

Coleman  found  dextrose,  cane  sugar,  glycerin,  and  lactose,  in 
small  amounts,  to  favor  nitrification,  and  in  some  cases  even  as 
much  as  1  per  cent,  of  dextrose  has  proved  beneficial.  This  con- 
clusion has  been  confirmed  by  numerous  other  workers.  Where 
larger  quantities  of  sugars  are  used  there  is  usually  a  disappearance 
of  nitrates.  This  is  probably  due  to  its  favoring  other  organisms 
which  produce  protein  from  the  nitrates  rather  than  interfering 
with  nitrification  or  accelerating  to  a  great  extent  denitrification. 
The  optimum  amount  of  organic  matter  for  most  rapid  nitrification 
varies  with  the  moisture  and  nature  of  the  soil.  Fischer  found 
even  peat  extract  to  favor  nitrification,  while  Niklewski  claims 
that  nitrification  occurs  in  solid  stable  manure  when  there  is  not 
much  liquid  manure  mixed  with  it,  and  that  on  the  first  day  nitrite 
bacteria  are  found  in  the  manure  coming  originally  not  from  the 


METABOLISM  223 

stock  but  from  the  straw,  particles  of  earth,  etc.,  that  stick  to  the 
manure.  These  bacteria  increase  in  number  until  at  the  end  of 
four  weeks  there  may  be  1000  per  gram  of  substance  associated 
with  these.  Hence,  we  may  conclude  that  the  absence  of  nitri- 
fication which  has  been  noted  by  various  workers  when  organic 
matter  is  present  may  be  due  to  some  of  the  following  factors: 
(1)  Excessive  quantities  of  soluble  organic  matter.  This  has  been 
repeatedly  found  to  be  the  case  where  excessive  quantities  of 
carbohydrates  have  been  added  to  the  media.  (2)  A  low  per- 
centage of  potassium  as  suggested  by  Renault.  (3)  The  physical 
and  chemical  properties  of  the  medium,  as  noted  by  Stevens  and 
Withers.  (4)  The  presence  of  organic  acid,  as  is  the  case  in  peats 
and  forest  soils.  In  this  condition  it  is  the  acid  reaction  which 
interferes  with  the  process  and  not  the  organic  matter  present. 
(5)  A  substance  may  be  toxic  when  tested  by  the  solution  method, 
whereas  in  the  soil  it  may  be  inert  or  actually  beneficial. 

Energy.— The  nitrifying  organisms  are  devoid  of  chlorophyll 
and  function  best  in  the  dark,  yet  they  synthesize  from  the  carbon 
dioxid  complex  organic  compounds.  The  energy  necessary  for 
this  synthesis  is  obtained  by  the  nitrosomonas  from  the  oxidation 
of  ammonia : 

2NH3     +     3O2     =     2HNO2     +     2H2O     +     157.6Cal. 

and  by  the  nitrobacter  from  the  oxidation  of  nitrous  acid: 

2HNO2     +     O2     =     2HNO3     +     36.6cal. 

Lafar  points  out  that  if  the  quantity  of  nitrogen  oxidized  per 
unit  of  time  be  taken  as  the  standard  for  measuring  the  chemical 
energy  of  these  organisms,  the  nitrosomonas  will  be  found  the 
mest  active  of  the  two.  From  this  fact  he  concludes  that  the 
conversion  of  the  trivalent  nitrogen  of  nitrous  acid  into  pentav- 
alent  nitric  nitrogen  requires  the  expenditure  of  a  greater  amount 
of  internal  force  than  is  needed  for  the  first  step  in  the  oxidation. 

Metabolism.— The  metabolism  of  these  organisms  has,  therefore, 
been  the  subject  of  considerable  study.  Winogradsky  early  sug- 
gested that  the  ammonium  carbonate  in  the  first  place  probably 
gives  rise  to  an  amid,  somewhat  similar  to  the  transforming  of 
ammonium  carbonate  into  urea: 


NH40  NH40  NH2 


^  \ 

\co  ->  Nco 


NH40/  NH2 

ammonium  ammonium 

carbonate  carbamate 


224  NITRIFICATION 

It  is  quite  likely  that  all  organic  compounds  are  first  trans- 
formed into  ammonia  by  other  organisms  before  they  are  nitrified. 
Demoussy  found  this  to  be  true  of  monomethylamin,  trimethylamin, 
anilin,  pyridin,  and  quinolin,  and,  according  to  Lohnis,  calcium 
cyanamid.  This  is  also  true  for  carbamid,  thiocarbamid,  uric 
acid,  acetamid,  anilin  sulphate,  methylamin  sulphate,  ammonium 
oxalate,  asparagin  and  ammonium  sulphate,  which,  with  the  excep- 
tion of  thiocarbamid  and  anilin  sulphate,  are  readily  transformed, 
according  to  Busley,  into  ammonia  by  other  bacteria  and  then 
nitrified.  Hence,  the  early  conclusion  reached  by  Winogradsky— 
that  pure  cultures  of  nitrifying  bacteria  are  incapable  of  nitrifying 
organic  nitrogen—  has  been  borne  out  by  other  investigators. 
Where  contrary  results  have  been  reported  it  has  been  due  to 
the  presence  of  other  organisms  by  which  the  nitrogen  has  been 
converted  into  ammonia  and  then  nitrified.  The  process  i's  cata- 
lyzed by  oxidizing  enzymes  which  must  be  specific  in  their  action, 
for  Omelianski  found  the  nitrifying  organisms  unable  to  oxidize 
mineral  compounds  such  as  sodium  sulphite  and  phosphite. 

Oxidation  in  this  case  cannot  be  regarded  as  being  of  a  violent 
nature  and  it  scarcely  seems  conceivable  that  the  nitrosomonas 
should  be  able  to  oxidize  ammonia  direct  to  nitrous  acid  without 
passing  through  intermediate  stages  of  oxidation.  Most  workers 
consider  it  probable  that  in  the  oxidation  of  the  ammonium  radical 
there  are  formed  certain  intermediate  substances  which  must  be 
regarded  as  more  or  less  hydroxylated  ammonium  radicals. 

Mulford,  in  a  study  of  the  bacterial  oxidation  of  aqueous  solu- 
tions of  ammonium  salts  on  experimental  filters  inoculated  from 
actively  nitrifying  sewage  filters  found  that  the  oxidation  proceeded 
in  a  series  of  stages  compatible  with  the  hypothesis  that  the  hydro- 
gen atoms  are  successively  hydroxylated  with  the  subsequent  elimi- 
nation of  water.  Hydroxylamin  salts  and  salts  of  hyponitrous 
acid  and  nitrous  acids  were  found  as  intermediate  compounds. 


/N 

\ 

hyponitrous 
/  acid 

H  H  H  O 

/  /  /   +  / 

N—  H      -»  N—  H        —  »  N—  OH  ---  »  N 

\  \  \  /     \ 

H  OH  OH  OH 

OH  nitrous 

ammonia     hydroxylamin  acid 

\N-OH 

\ 
dihydroxylamin  OH 

trihydroxyl- 
amin  acid 


MORPHOLOGY  225 

There  are,  however,  two  serious  objections  to  these  conclusions: 

(1)  It  is  not  evident  that  these  initial  changes  noted  by  Mulford 
were  due  to  the  nitrifying  organisms,  as  a  mixed  culture  was  used; 

(2)  Boullanger  and  Massol  found  that  while  the  nitrous  organism 
accommodates  itself  to  all  ordinary  carbonates,  it  does  not  attack 
hydroxylamin  hydrochlorid. 

The  majority  of  workers  have  reported  a  loss  of  nitrogen  in  the 
nitrification  process,  there  never  being  the  theoretical  yield  of  100 
per  cent,  of  the  ammonia  transformed  into  nitrous  acid,  but  this 
may  be  due  to  side  reactions  Lafar  considers  that  the  loss  may 
be  due  to  the  reaction  of  the  nitrous  acid  on  the  undecomposed 
ammonia  in  accordance  with  the  equation : 

N2O3     +     2NH3      =     3H2O     +     2N2 

The  whole  subject  of  the  metabolism  of  nitrifiers  is  indefinite 
and  in  need  of  careful  investigation  using  the  latest  refined  methods. 
The  only  fact  that  does  seem  to  be  well  established  is  that  the 
process  of  nitrification  goes  in  two  stages  from  ammonia  to  nitrous 
acid  and  from  nitrous  acid  to  nitric  acid.  That  these  two  steps 
are  due  to  two  classes  of  organisms  is  the  claim  of  most  investi- 
gators. However,  Kaserer  considers  that  there  is  an  organism, 

B.  nitrator,  which  can  oxidize  ammonia  direct  to  nitric  acid,  the 
reactions  being  as  follows: 

NH3     +     H2CO3     +     O2      =     HNO3     +     H2O     +     CH2O     —     41  Cal. 
CH2O     +     O2     =  •  H2CO3     +     132  Cal. 

It  is  interesting  to  note  that  the  reaction  catalyzed  by  the  nitri- 
fying ferments  are  similar  to  reactions  catalyzed  by  ultraviolet 
rays.  Gaudechon  exposed  solutions  at  temperatures  of  35°  to  50° 

C.  for  from  three  to  nine  hours  at  a  distance  of  3  to  6  cm.  from  a 
lamp  of  110  watts.     Under  these  conditions  the  ultraviolet  rays 
oxidized  solutions  of  ammonia  in  the  presence  of  oxygen  to  nitrites. 
Nitrates  were  in  no  case  formed.     Ammonium   salts  were  also 
oxidized  to  nitrites,  the  reaction  being  slower  in  the  case  of  the 
sulphates  and  chlorids  than  the  carbonates.     Urea  was  first  con- 
verted into  ammonia  and  then  into  nitrites.     Other  organic  nitro- 
gen compounds,  for  example,  ethyl-  and  methylamin,  guanidin, 
hydroxylamin,  acetamid,  and  acetonitril  behaved  similarly. 

Morphology.— Winogradsky  described  two  varieties  of  the  organ- 
isms capable  of  changing  ammonia  to  nitrites.  One  of  these  in 
several  species  was  found  in  all  the  soils  of  the  Old  World  (Asia, 
Africa  and  Europe)  and  is  known  as  nitrosomonas.  The  second 
is  peculiar  to  the  soil  of  the  New  World  and  has  received  the  name 
of  nitrosococcus. 

He  described  a  single  species  of  the  nitrosomonas  from  European 
soils,  namely,  Nitrosomonas  europcea.  This  organism  is  provided 


226  NITRIFICATION 

with  a  single  short  flagellum  and  in  the  early  stages  of  the  culture  it 
exhibits  active  powers  of  locomotion.  It  appears  as  short  rods 
••SI. 2-1. 8  M  long  and  0.9-1.0  /*  broad.  The  cells  of  Nitrosomonas 
javanica  obtained  from  the  Botanical  Garden  at  Buitenzorg,  near 
Batavia,  are  globular  and  only  attain  a  diameter  of  0.5-0.6  ju,  but 
they  have  a  long  flagellum,  at  times  measuring  as  much  as  30  //. 
Those  obtained  from  Tokio  soil  ( Nitrosomonas  japonica)  and  from 
Africa  (Nitrosomonas  africana),  are  very  similar  to  the  European 
species,  differing  only  in  that  they  are  somewhat  smaller. 

Observations  by  Burri  and  Stutzer  on  impure  cultures  in  mineral 
media  led  them  to  believe  that  there  was  a  difference  in  oxidizing 
powers  in  organisms  derived  from  different  sources.  By  this  means 
they  distinguished  five  classes  from  German  and  one  from  African 
soil. 

Joshi  has  recently  described  a  new  species  from  the  soils  of 
India  which  differ  morphologically  from  others  hitherto  described. 

The  different  species  show  a  variation  in  sensitiveness  to  heat. 
Beddies  found  one  species  to  live  for  one  minute  in  steam  at  a 
temperature  of  100°.  The  other  two  were  more  sensitive  but 
survived  for  several  minutes  in  dry  heat  of  80°  to  100°  C. 

The  genus,  nitrococcus,  found  in  the  New  World  do  not  possess 
cilia  nor  do  they  form  zooglea.  The  one  obtained  from  Quito 
(Ecuador)  is  a  coccus  1.5-1.7  ju  in  diameter.  The  species,  Nitro- 
sococcus  braziliensis,  obtained  from  Brazil  soil  is  much  larger,  being 
2  n  in  diameter. 

The  nitromonas  or  nitrobacter  differ  from  those  already  described 
in  physiological  properties  in  that  they  oxidize  nitrites  into  nitrates. 
Morphologically,  they  differ  in  being  smaller  and  more  slender. 
They  are  elongated,  oval,  mostly  pear-shaped,  0.5  n  in  length  and 
0.15-0.25  ju  in  breadth.  In  liquid  cultures  they  develop  a  thin 
mucinous  skin  which  adheres  firmly  to  the  walls  of  the  vessel. 

From  the  variation  in  sensitiveness  to  heat,  Beddies  isolated 
four  forms  of  nitrobacter,  one  of  which  was  capable  of  resisting 
the  action  of  steam  at  100°  C.  for  two  minutes.  But  Burri  and 
Stutzer's  comparative  experiments  with  nitric  organisms  derived 
from  different  localities  showed  no  essential  difference  in  physio- 
logical action. 

Neither  nitrosomonas  nor  nitromonas  have  been  observed  to 
form  spores,  but  their  resistance  to  drying  and  to  heat,  as  shown  by 
Beddies,  makes  it  appear  possible  that  some  species  may  form  spores. 

Influence  of  Moisture.— Long  before  the  process  of  nitrification 
was  known  to  be  due  to  microorganisms,  the  underlying  principles 
governing  the  speed  of  the  reaction  had  been  investigated  nation- 
ally by  France,  Germany  and  Sweden.  Among  other  things,  they 
had  learned  that  there  must  be  a  certain  proportion  of  water,  and, 
in  order  that  the  maximum  vield  of  nitrates  be  obtained,  that  this 


INFLUENCE  OF  MOISTURE  227 

must  be  diminished  as  the  soil  becomes  richer  in  nitrates.  As  early 
as  1887  Deherain  found  that  the  most  active  nitrification  took  place 
when  the  soil  was  allowed  to  become  partially  dry  between  the 
applications  of  water,  and  later  he  found  that  there  was  a  rela- 
tionship between  the  speed  of  nitrification  and  the  moisture  con- 
tent of  fallow  soil,  the  nitrification  increasing  with  the  water. 
Boussingault  taught  that  when  soils  contain  as  much  as  60  per 
cent,  of  water  they  lose  in  a  few  weeks  the  greater  part  of  their 
nitrates.  This  teaching  gave  rise  to  the  general  belief  that  deni- 
trification  may  take  place  to  a  great  extent  in  soils,  but  recent 
work  has  amply  demonstrated  that  it  is  only  extremely  abnormal 
conditions  where  this  becomes  an  important  factor. 

Deherain  and  Demoussy  found  that  the  bacterial  action  of  a 
soil  was  at  its  maximum  when  a  rich  soil  contained  17  per  cent, 
of  water,  but  that  it  decreased  if  the  proportion  of  water  fell  to 
10  per  cent,  or  rose  to  25  per  cent.  With  soils  less  rich  in  humus 
a  somewhat  higher  proportion  of  water  was  necessary  to  retard 
oxidation  to  any  marked  degree. 

The  optimum  moisture  content  for  nitrification,  according  to 
Deherain,  is  25  per  cent.  An  insufficient  supply  of  moisture 
checked  both  nitrification  and  nitrogen  fixation.  This  occurred 
when  the  water  had  been  reduced  to  16.5  per  cent.  This,  however, 
would  vary  with  the  soil,  for  Schlosing  found  bacterial  activity 
less  in  fine-grained  soils  than  in  lighter,  coarse-grained  soils.  In 
order  that  nitrification  be  equally  active  in  both  light  and  heavy 
soils,  the  latter  must  have  a  higher  percentage  of  water  than  the 
former,  a  difference  in  moisture  content  of  soil  of  1  per  cent.,  accord- 
ing to  Dafert  and  Bellinger,  being  sufficient  to  produce  a  marked 
change  in  the  oxidation  going  on  in  the  soil. 

Fraps  found  that  the  number  of  nitrifying  organisms  in  a  soil 
varies  with  the  moisture  and  that  their  activity  was  periodic,  rapid 
nitrification  being  preceded  and  followed  by  periods  of  less  activity. 
Later  he  found  nitrification  to  be  at  its  height  in  soil  containing 
55.6  per  cent,  of  its  water-holding  capacity.  Excessive  quantities  of 
water  practically  stopped  nitrification  and  were  much  more  injur- 
ious than  too  small  a  quantity.  The  water  requirements,  however, 
varied  considerably  with  the  soil.  Coleman's  work  with  a  loam 
soil  showed  nitrification  to  be  most  active  when  the  soil  contained 
16  per  cent,  of  water.  It  was  greatly  retarded  when  the  water 
content  was  reduced  to  10  per  cent,  or  increased  to  26  per  cent. 

Patterson  and  Scott's  work  is  interesting  in  that  they  found 
nitrification  to  be  inactive  in  sand  and  clay  soils  which  still  con- 
tained about  three  times  as  much  moisture  as  in  their  average  air- 
dry  condition.  At  the  lower  limits  of  moisture  less  water  starts 
nitrification  in  sand  than  in  clay.  At  the  higher  limits  of  moisture 
less  water  stops  nitrification  in  sand  than  in  clay,  while  the  opti- 


228  NITRIFICATION 

mum  amount  of  water  probably  varies  for  each  soil;  it  is  higher 
for  clay,  yet  for  both  soils  it  lies  within  the  range  of  from  14  to 
18  per  cent.  A  rise  above  the  optimum  amount  of  water  is  more 
harmful  than  an  equal  fall  below  it. 

The  work  of  the  Utah  Experiment  Station  demonstrated  that 
the  application  of  irrigation  water  to  a  soil  has  a  distinct  beneficial 
effect  upon  nitrification,  being  greatest  where  15  inches  of  water 
were  applied  when  the  nitric  nitrogen  formed  amounted  to  28.5 
pounds  per  acre-foot  of  soil.  The  greatest  benefit  per  inch  of 
water,  however,  was  obtained  where  only  7.5  inches  of  water  were 
applied,  resulting  in  3.8  pounds  of  nitric  nitrogen  per  inch  of  water, 
while  where  15  inches  were  applied  it  was  1.1  pounds  of  nitric  nitro- 
gen per  inch  of  water  applied,  and  when  25  inches  of  water  were 
applied  to  the  soil  the  nitric  nitrogen  produced  was  only  0.7  pound. 

Mtinter  and  Robson  found  that  hornmeal  decomposed  more 
rapidly  in  dry  sandy  soil  than  in  clay  or  loam,  whereas  with  higher 
moisture  content  there  was  little  difference.  Ammonia  sulphate 
transformation  increased  with  a  higher  water  content.  The  best 
nitrate  formation  from  hornmeal  occurred  in  sandy  soils.  In  clay 
and  loam  it  was  best  with  a  medium  water  content.  Sharp  found 
that  the  water  content  most  favorable  for  ammonification  was 
not  the  optimum  condition  for  nitrification.  The  former  was 
most  rapid  with  a  25  per  cent,  water  content  and  was  not  markedly 
affected  by  3  per  cent,  differences.  Nitrification  was  at  its  maxi- 
mum when  the  soil  contained  19  per  cent,  of  water.  When  it  was 
increased  to  25  per  cent,  the  rate  of  nitrification  was  decreased 
50  per  cent. 

McBeth  and  Smith  found  a  slight  variation  in  the  number  and 
nitrifying  powers  of  soil,  depending  upon  the  moisture  content. 
However,  Gainey  considers  that  among  the  factors  controlling  the 
bacterial  activity  of  a  soil  the  available  moisture  probably  plays 
a  leading  part.  But  the  author  has  reported  results  which  indicate 
that  the  nitrous  nitrogen  content  of  a  soil  is  independent  of  the 
irrigation  water  applied  up  to  37.5  inches  a  year.  Results  recently 
published  by  the  Utah  Experiment  Station  clearly  demonstrate 
that  the  influence  exerted  by  water  upon  ammonifying,  nitrifying, 
and  nitrogen-fixing  activities  of  the  soil  varies  greatly  with  the 
organic  matter  in  the  soil  and  is  much  more  marked  in  effect  on 
soils  recently  manured  than  on  those  which  have  received  no 
manure. 

From  the  literature  cited  it  may  be  seen  that  the  nitrifying  power 
of  the  soil  is  a  function  of  the  moisture  content  of  the  soil,  and 
that  the  optimum  varies  with  the  physical  and  possibly  with  the 
chemical  properties  of  the  soil.  Recent  work  at  the  Utah  Experi- 
ment Station  shows  a  close  correlation  between  the  nitrifying 
powers  of  a  soil  and  its  water-holding  capacity  and  varies  only 


INFLUENCE  OF  MOISTURE 


229 


slightly  with  the  physical  properties  of  a  soil.  Twenty-two  soils 
varying  widely  in  physical  properties  yielded  maximum  nitrification 
when  the  soil  contained  from  50  to  60  per  cent,  of  their  water- 
holding  capacity,  as  indicated  in  Fig.  30.  Furthermore,  the  opti- 
mum moisture  content  for  maximum  nitrification  is  correlated 


0       10     20      30      40      X      60      70      80      90     IOC 


FIG.  32. — Average  percentages  of  nitric  nitrogen  produced  in  soil  receiving  various 
quantities  of  water.  The  quantity  produced  at  60  per  cent,  is  taken  as  100;  on  the 
ordinate  is  given  the  per  cent  of  nitric  nitrogen  formed,  whereas  on  the  abscissa  is 
given  water  applied  as  per  cent,  of  water-holding  capacity. 


with  the  other  soil  constants  with  a  set  of  equations  similar  to 
those  given  for  ammonification,  page  201.     Thus, 


Mn  =  .55      C 

Mn  =  .8525E  +  11.55 

Mn  =  1.472    W  +  11.55 

Mn  =  2.163    H  +  11.55 


230  NITRIFICATION 

Mn  is  written  for  per  cent,  of  water  for  maximum  nitrification, 
C  for  moisture  capacity  as  defined  by  Hilgard,  W  for  wilting  co- 
efficient, E  for  moisture  equivalent,  and  H  for  hygroscopic 
coefficient. 

Temperature.— The  temperature  is  a  factor  which  controls  in  a 
great  measure  the  quantity  of  nitrates  produced  in  unit  time. 
Schlosing  found  nitrification  very  slow  at  7.5°  C.,  quite  marked  at 
11°,  reached  its  maximum  at  37°,  and  ceased  entirely  at  55.° 
Deherain  found  nitrification  almost  ceased  at  5°  C.  and  begins  very 
slowly  in  soils  which  have  been  frozen,  yet  Conn  found  the  freezing 
of  soil  increases  its  nitrifying  powers.  These  temperatures  are 
questioned  by  some,  for  example  Warington  states  that  he  was 
unable  to  start  nitrification  at  40°  C. 

Hutchinson  gives  the  optimum  temperature  for  nitrification  in 
Pusa  soil  at  35°  C.  No  nitrates  were  formed  at  40°,  nor  did  nitri- 
fication take  place  in  soil  which  had  been  kept  at  40°  C.  when  its 
temperature  was  afterward  reduced  to  30°  C.  These  apparent 
contradictions  may  be  due  to  different  strains  of  the  organisms 
varying  in  sensitiveness  to  heat.  Beddies  isolated  four  stable 
forms  of  nitric  and  three  of  nitrous  ferments.  One  of  the  nitric 
forms  was  capable  of  resisting  the  action  of  steam  at  100°  C.  for 
two  minutes  and  one  of  the  nitrous  bacteria  lived  for  one  minute 
in  steam  at  the  same  temperature.  The  other  two  nitrous  ferments 
could  not  withstand  steam  but  survived  for  several  minutes  in  a 
dry  heat  of  80°  to  100°  C.  Moreover,  Bazarewski  found  the  most 
favorable  temperature  for  nitrification  in  soils  to  be  between  25°  and 
27° "C.,  or  about  10°  C.  lower  than  in  pure  cultures  in  artificial 
media. 

King,  in  his  work,  found  that  there  was  1.26  times  as  much 
nitric  nitrogen  formed  at  9°  C.  as  at  1°  C.,  2.76  times  as  much  at 
20°,  and  6.24  times  as  much  at  35°,  as  at  1°.  The  significance  of 
these  figures  is  brought  out  more  fully  when  we  examine  the  amounts 
of  nitric  nitrogen  obtained  in  some  cases.  At  1°  C.  there  were 
formed  120  pounds  per  acre;  at  9°,  150  pounds  per  acre;  at  20°,  329 
pounds  per  acre;  while  at  35°  there  were  formed  747  pounds  per  acre. 

Light  Rays.— The  nitrifying  organisms  are  heat-loving  and  light- 
avoiding.  They  are  dependent  on  the  heat  of  the  earth  or  of  the 
sun,  but  they  carry  on  their  activities  best  in  the  absence,  of  sunlight. 
Direct  sunlight,  partly  due  to  the  coagulation  of  the  bacterial  col- 
loids by  the  rays  of  the  ultraviolet  light,  soon  proves  fatal  to  them. 

Aeration  and  Cultivation.— The  nitrifying  bacteria  are  all  aerobic; 
hence,  nitrification  is  best— other  things  being  equal— in  a  well- 
aerated  soil.  This  is  illustrated  by  the  work  of  Schlosing  who 
exposed  soil  for  four  months  to  an  atmosphere  containing  different 
percentages  of  oxygen.  Soil  which  contained  1.5  per  cent,  of 
oxygen  yielded  45.7  mg.  of  nitric  nitrogen,  that  containing  6  per 


CROP  AND  FALLOW  231 

cent  yielded  95.7,  that  containing  11  per  cent,  yielded  132.5  mg., 
whereas  that  containing  16  per  cent,  of  oxygen  yielded  246.6  mg. 
of  nitric  nitrogen. 

Plummer  found  there  to  be  an  optimum  mixture  of  carbon 
dioxid  and  oxygen  for  the  best  production  of  nitrates.  This  he 
found  to  be  one  containing  from  35  to  60  per  cent,  of  oxygen.  But 
Hutchinson  found  complete  nitrification  of  ammonium  sulphate  took 
place  under  semi-anaerobic  conditions  in  which  no  nitrification  of 
oil  cake  occurred. 

Stirring  and  pulverizing  the  soil  is,  therefore,  of  great  importance, 
as  further  shown  by  the  experiments  of  Deherain.  A  number  of 
pots  were  filled  with  soil.  Part  of  them  were  allowed  to  stand 
undisturbed,  while  the  others  were  poured  out  upon  the  floor  and 
frequently  stirred.  Those  stirred  invariably  contained  from  ten 
to  forty  times  as  much  nitrates  as  did  the  unstirred. 

The  work  of  King  also  shows  that  the  stirring  of  the  soil  affe/cts 
nitrification.  He  further  found  land  plowed  in  the  fall  contained 
a  different  amount  of  nitrates  than  did  the  unplowed  land,  the 
difference  being  apparent  throughout  the  following  summer. 

Crop  and  Fallow.— Even  as  early  as  1855  the  work  at  Rothamsted 
had  demonstrated  that  the  beneficial  effects  of  fallowing  lies  in 
the  increase  brought  about  in  the  available  nitrogen  compounds 
of  the  soil.  Deherain  and  Demoussy's  work  indicated  that  there 
is  a  larger  production  of  nitrates  in  fallow  than  in  cropped  soils, 
and  Pfeiffer  considers  fallowing  an  extreme  form  of  soil  robbery, 
for  he  found  that  it  promotes  the  activity  of  the  soil  organisms,  and 
hence  hastens  the  exhaustion  of  the  nitrogen  supply.  But,  as  it 
is  so  clearly  pointed  out  by  Warington,  these  results  may  not  hold 
in  a  dry  climate  or  during  dry  seasons;  for  here  bare  fallow  may 
not  necessitate  this  loss  and  much  is  to  be  gained  by  its  practice. 
But  it  must  always  be  borne  in  mind  that  if  there  be  sufficient 
moisture  the  loss  may  be  great.  For  instance,  Schneidewind, 
Meyer  and  Munter  record  a  loss  in  fallow  plats  of  85.5  pounds 
per  acre,  which  even  exceeded  the  nitrogen  removed  by  the  growing 
plant  on  the  cropped  soil. 

On  the  other  hand,  McBeth  and  Smith  claim  that  plats  con- 
tinuously cropped  to  alfalfa,  potatoes,  oats,  and  corn  all  show  a 
higher  nitrifying  power  than  do  corresponding  fallow  plats  and 
that  the  stimulating  effect  of  crop  production  on  the  nitrifying 
power  of  a  soil  is  most  marked  in  alfalfa  soil.  This  is  in  keeping 
with  the  recent  findings  of  Welbel,  but  is  contrary  to  the  findings 
of  many  other  investigators,  for  Heinze  found  fallow  to  increase 
the  pectin,  cellulose  and  humus  fermenters  and  also  the  ammoni- 
fiers,  nitrifiers  and  Azotobacter.  Russel  finds  that  late  summer 
fallow  land  is  richer  in  nitrates  than  is  cropped,  even  after  allowing 
for  the  nitrogen  taken  up  by  the  crop;  and  Heinze  shows  that 


232  NITRIFICATION 

repeated  cultivation  of  fallow  soil  increased  the  number  of  organ- 
isms in  the  soil,  while  Hiltner  maintains  that  no  nitrification  occurs 
in  soils  where  legumes  are  growing  vigorously  and  fixing  large 
quantities  of  nitrogen.  This  latter  view,  however,  is  the  extreme, 
as  is  shown  by  much  of  the  literature  on  the  subject. 

Welbel  and  Winkler  found  that  fallow  not  only  increased  the 
assimilable  nitrogen,  but  also  the  available  phosphoric  acid  of 
the  soil,  and  that  the  increased  yield  of  wheat  after  fallow  is  due 
to  these  factors.  But  Bychikhin  and  Skalski  point  out  that  fall 
fallow  is  even  more  wasteful  of  soil  nitrates  than  is  summer  fallow, 
for  here  the  excessive  rains  wash  the  soluble  nitrates  from  the  soil 
as  fast  as  formed.  The  cultivating  of  fallow  further  increases  the 
nitrate  content,  as  was  shown  by  Richardson.  Nitrification  is 
related  to  fallow  and  crop,  as  may  be  seen  from  the  following  results 
obtained  by  the  author: 

Milligrams  Milligrams 

nitric  nitrogen  nitrogen 

formed.  fixed. 

Cultivated 4.16  14.28 

Virgin  soil 2.09  6.99 

Wheat  soil 4.00  11.83 

Alfalfa  soil      ..........  2.25  12.24 

Fallow  soil,  potato  fallow,  etc.        ...  6.22  22.88 

The  results  reported  under  milligrams  of  nitrogen  fixed  indicate 
that  in  an  arid  soil  the  increased  nitrogen  fixation  in  a  fallow  soil 
more  than  offsets  the  loss  of  nitrates,  even  though  rapidly  formed, 
for  little,  if  any,  would  be  lost  in  the  drainage  waters.  These 
results  have  recently  been  confirmed  by  Reed  and  Williams.  More- 
over, the  number  of  organisms  in  the  soil  and  the  rapidity  of  the 
bacterial  activity  within  the  same  is  going  to  vary  greatly  with 
the  thoroughness  and  time  of  cultivation,  as  shown  by  Deherain, 
Neish,  King  and  Whitson,  Chester  and  Quiroga,  while  the  number 
and  activity  of  the  organisms  in  the  soil  may  in  a  degree  determine 
the  speed  with  which  the  water  evaporates  from  a  soil. 

The  work  at  the  Rothamsted  station  early  demonstrated  that 
the  nitrates  in  the  drainage  water  from  the  various  plats  varied 
greatly,  depending  upon  the  crop  growing  upon  the  soil,  thus  indi- 
cating a  relationship  between  the  available  nitrogen  in  a  soil  and 
crop  growing  upon  the  soil.  Since  that  time  many  experiments 
have  confirmed  this  conclusion.  Furthermore,  King  and  Whitson 
found  22  per  cent,  more  nitrogen  developed  from  soil  after  clover 
than  from  soil  after  corn,  and  13  per  cent,  more  than  after  oats. 
Later  work  by  them  showed  that  there  are  greater  quantities  of 
nitrates  throughout  the  entire  season  in  soil  under  corn  or  potatoes 
than  in  soil  under  clover  and  oats.  Stewart  and  Greaves  found 
that  different  plants  show  a  marked  difference  in  their  demands 


CROP  AND  FALLOW  233 

upon  the  nitrate  content  of  the  soil,  there  being  a  steady  decrease 
in  the  concentration  of  the  nitrate  content  of  potato  and  corn 
lands  as  the  season  progressed,  while  that  of  fallow  and  alfalfa 
remained  practically  constant,  the  nitrate  content  of  the  latter 
being  uniformly  low  through  the  season.  According  to  Lyon  and 
Bizzell,  soil  that  had  produced  alfalfa  for  five  years  was  higher  in 
nitrates  than  soil  that  had  grown  timothy  during  the  same  period. 
Furthermore,  the  former  nitrified  ammonium  sulphate  more  readily 
than  did  the  latter. 

Brown  found  that  the  rotation  of  crops  caused  an  increase  in 
number  of  organisms  in  a  soil,  also  greater  ammonifying,  nitrifying 
and  nitrogen-fixing  powers  than  continuous  cropping  to  either  corn 
or  clover.  Furthermore,  the  crop  on  the  soil  at  time  of  sampling 
was  of  more  importance  from  the  bacterial  viewpoint  than  the 
previous  crop.  However,  the  preceding  crop  has  a  marked  effect 
upon  the  nitrate  content  of  the  soil,  as  is  seen  from  the  work  of 
Lyon  and  Bizzell,  where  plats  that  had  been  planted  to  certain 
crops  were  kept  bare  of  vegetation  in  the  early  part  of  the  growing 
season  of  1911.  Nitrate  determinations  of  the  soil  were  made  and 
the  nitrate  present  showed  a  distinct  and  characteristic  relationship 
to  the  nitrate  content  found  under  the  several  varieties  of  plants 
previously  grown  upon  the  soil.  Later  they  showed  that  alfalfa 
soil  nitrified  more  rapidly  than  timothy  soil,  both  in  the  soil  on 
which  the  crops  had  been  grown  continuously  and  in  that  from 
which  they  had  been  removed  and  the  soil  kept  bare  for  two  seasons. 
The  author  has  shown  that  the  nitrifying  powers  of  alfalfa  soil, 
while  slightly  higher  than  that  of  virgin  soil,  is  very  low  when 
compared  with  either  wheat  or  potato  and  fallow  soil.  Further- 
more, the  extensive  work  which  has  been  conducted  at  the  Utah 
Experiment  Station  demonstrates  that  there  is  a  very  pronounced 
relationship  between  the  crop  growing  upon  a  soil  and  its  nitrate 
content.  However,  in  this  work  the  nitrate  content  of  the  alfalfa 
and  oat  soil  is  very  low,  while  that  of  potatoes  and  fallow  is  high, 
and  we  find  the  nitrifying  powers  of  alfalfa  and  potato  soil  high  as 
compared  with  fallow. 

Nitric  nitrogen  Nitrifying 

Crop.  in  soil.  powers. 

Fallow1 100  100 

Alfalfa 36  148 

Oats 36  103 

Corn 33  77 

Potatoes 99  21 

Hence,  we  can  conclude  that  alfalfa  not  only  feeds  closer  upon 
the  soluble  nitrates  of  the  soil  but  also  makes  a  much  greater  drain 

1  Fallow  taken  as  100. 


234  NITRIFICATION 

upon  the  insoluble  nitrogen  of  the  soil  by  increasing  its  nitrifying 
powers. 

Season.— The  season  of  the  year  has  a  marked  influence  upon 
the  bacterial  activities  of  the  soil,  but  it  is  not  necessarily  corre- 
lated with  the  nitrate  content  of  the  soil.  Schlosing  found  the 
nitrates  in  the  drain  water  from  both  manured  and  unmanured  soil 
high  in  spring,  as  compared  with  midsummer,  fall,  or  winter,  thus 
confirming  the  results  obtained  at  the  Rothamsted  station.  Shutt 
reports  nearly  five  times  the  quantity  of  nitrates  in  fallow  and 
cropped  soil  during  June  as  during  November.  He  does,  however, 
find  more  during  June  than  during  May.  The  exact  season  of 
the  year  at  which  the  maximum  nitrate  content  is  reached  will 
vary  with  a  number  of  factors,  chief  among  which  is  the  kind  of 
crop  growing  on  the  soil,  for  King  and  Whitson  found  that  the 
nitrates  in  the  surface  foot  start  in  the  spring  comparatively  low 
and  increase  rapidly  until  June  1  on  clover  and  oat  ground,  and 
until  July  on  corn  and  potato  ground.  From  these  dates  they  fall 
more  or  less  rapidly  and  the  work  at  the  Utah  Station  demonstrates 
conclusively  that  there  is  a  seasonal  variation,  depending  upon 
temperature,  crop  and  quantity  of  irrigation  water  applied  to 
the  soil. 

Moreover,  Andre  has  shown  that  the  insoluble  nitrogenous 
compounds  of  the  surface  soil  are  largely  transformed  into  soluble 
compounds  during  the  summer,  and  these  are  widely  diffused 
through  the  deeper  layers  of  soil  during  the  winter,  so  that  in  the 
spring  the  lower  layers  of  soil  contain  more  soluble  nitrogen  than 
the  .surf  ace  soil.  At  the  end  of  summer,  however,  the  distribution 
is  quite  uniform.  This  finding  has  been  amply  verified  by  the 
results  reported  by  Stewart  and  Greaves,  Welbel,  Jensen,  and 
Lyon  and  Bizzell.  The  results  will  vary,  however,  with  different 
soils,  as  shown  by  Russell  who  reports  the  fluctuations  in  nitrates 
more  marked  on  loams  than  on  clays  or  sands.  Moreover,  he 
found  the  bacterial  activities  much  greater  in  early  summer  than 
later. 

Moll  even  goes  so  far  as  to  claim  from  his  work  that  the  season 
of  the  year  is  the  principal  factor  in  determining  the  biochemical 
transformation  in  a  soil,  and  Heinze  found  that  the  number  of 
organisms  in  a  soil  was  highest  in  the  summer  months  and  lowest 
in  the  fall  and  spring.  As  already  pointed  out,  the  highest  nitri- 
fying power  of  a  soil  is  not  necessarily  correlated  with  the  highest 
nitrate  content.  The  latter  is  highest  in  spring  or  early  summer, 
while  Vogel  found  the  former  to  be  highest  in  October  and  Novem- 
ber, after  which  there  was  a  falling  off  until  April,  when  it  rose 
again,  but  not  so  high  as  in  autumn.  This  corresponds  fairly  well 
with  the  findings  of  Green,  for  the  ammonifying  powers  of  the 


QUANTITY  OF  NITRATES  FORMED  235 

soil.  These  findings,  however,  are  contrary  to  those  of  Wojtkie- 
wicz,  who  found  the  maxiihum  number  of  organisms  to  occur  in 
soil  during  the  spring  and  the  minimum  in  the  winter.  He  also 
notes  a  correlation  between  bacteria  present  and  the  amount  of 
nitrates  in  the  soil. 

Climate  influenced  the  nitrifying  powers  of  the  soil,  and  Hilgard 
taught  that  the  nitrifying  powers  of  the  arid  soils  are  superior  to 
those  of  the  humid  soils,  but  the  extensive  work  by  C.  B.  Lipman, 
both  by  laboratory  and  field  experiments,  in  which  soils  have  been 
transported  from  humid  to  arid  districts,  and  vice  versa,  has  shown 
just  the  opposite  to  be  true— namely,  that  the  biological  activities 
of  a  soil  are  more  pronounced  under  humid  than  under  arid 
conditions. 

Quantity  of  Nitrates  Formed.— The  quantity  of  nitrates  produced 
in  a  given  soil  varies  with  all  of  the  factors  which  have  been  con- 
sidered; hence,  any  results  obtained  must  be  interpreted  with 
this  in  mind.  The  greatest  rate  of  nitrification  noted  by  Warington, 
when  working  with  an  ordinary  arable  soil  from  the  Rothamsted 
farm,  yielded  0.588  parts  of  nitrogen  per  million  of  air-dried  soil 
a  day.  Similar  soil  supplied  with  ammonium  chlorid  nitrified 
about  0.924  parts  per  million  in  the  same  time. 

Lawes  and  Gilbert,  working  with  the  far  richer  Manitoba  soils 
and  with  a  higher  temperature,  obtained  in  two  cases  (soils  from 
Selkirk  and  Winnipeg)  average  daily  rates  of  nitrification  of  0.7 
parts  of  nitrogen  per  million  during  three  hundred  and  thirty-five 
days,  the  rates  during  the  early  portion  of  this  period  being  as  high 
as  1.03,  1.24,  1.36  and  1.72  per  million. 

Deherain,  working  with  a  soil  containing  0.16  per  cent,  of  nitro- 
gen, obtained  daily  rates  of  nitrification  varying  from  0.71  to  1.09 
per  million  in  ninety  days.  Working  with  a  richly  manured  soil 
containing  0.261  per  cent,  of  nitrogen,  he  obtained  a  maximum 
daily  rate  of  nitrification  during  forty  days  of  1.48  of  nitrogen 
per  million  of  soil. 

At  times  the  difference  in  nitrification  noted  in  different  soils 
may  be  due  to  a  difference  in  physiological  efficiency  of  the  nitrify- 
ing ferment,  as  Marcille  compared  the  nitrifying  powers  of  three 
different  soils  and  found  that  the  poorest  yielded  an  organism 
nitrifying  less  rapidly  than  the  others.  Some  soils  nitrify  ammonia 
more  readily,  while  others  nitrify  cotton-seed  meal  more  rapidly. 
This  must  be  due  to  differences  in  the  metabolism  of  the  organism 
found  in  the  various  soils. 

Hutchinson  considers  this  variation  at  times  due  to  toxins  which 
develop  under  anaerobic  conditions  produced  by  water  saturation. 
Subsequent  aeration  removes  the  toxic  condition  and  the  formation 
of  nitrates  takes  place.  He  also  found  copper  had  a  decided 


236  NITRIFICATION 

influence  in  neutralizing  the  toxic  action.  Several  other  observers, 
including  Greig-Smith  and  Bottomley,  claim  to  have  found  soluble 
bacteria  toxins  in  soil.  Russell  and  Hutchinson,  on  the  other  hand, 
obtained  wholly  negative  results  and  concluded  that  soluble 
bacterio-toxins  are  not  normal  constituents  of  soils,  but  must 
represent  unusual  conditions  wherever  they  occur.  But,  as  pointed 
out  by  Russell  the  possibility  of  the  existence  of  toxins  soluble  in 
water  still  remains. 

Loss  of  Nitrates.— The  loss  of  nitric  nitrogen  from  a  soil  may  be 
either  great  or  small,  depending  upon  certain  factors,  the  more 
important  of  which  are  as  follows: 

1.  The  rapidity  of  nitrification.     Nitric  nitrogen  may  be  pro- 
duced in  some  soils  so  rapidly  that  even  luxuriant  vegetation  will 
not  remove  it  as  fast  as  formed,  whereas  in  another  soil  it  may  be 
formed  so  slowly  that  it  will  not  suffice  for  even  meager  growth. 
The  loss  in  the  first  case  may  be  very  large,  while  that  of  the  second 
would  be  nearly  zero. 

2.  The  nature  of  the  soil.     A  tight  soil,  other  things  being  equal, 
would  retain  the  nitric  nitrogen  to  a  greater  extent  than  would 
a  loose  porous  soil,  and  a  deep  soil  than  a  shallow  soil. 

3.  The  amount  and  distribution  of  rainfall.     All  other  condi- 
tions being  equal,  thirty  inches  of  precipitation  throughout  the 
year  would  remove  more  nitric  nitrogen  from  the  soil  than  would 
fifteen  inches  similarly  distributed.     But  if  the  fifteen  inches  came 
within  a  short  period,  while  the  thirty  was  distributed  through- 
out the  year,  the  fifteen  inches  of  rainfall  under  these  conditions 
may  remove  more  nitric  nitrogen  from  'the  soil  than  would  the 
thirty. 

4.  The  rapidity  with  which  the  nitric  nitrogen  is  removed  by 
the  growing  crop.     Alfalfa,  oats  and  wheat  are  heavy  feeders  upon 
nitric  nitrogen  and  in  most  soils  remove  it  as  fast  as  formed.     Hence, 
little  is  left  to  be  washed  out  by  the  drainage.     Moreover,  crops 
such  as  these  rapidly  remove  the  water  from  the  soil  and  hence 
diminish  the  drainage  from  such  soils.    Moreover,  crops  growing 
during  the  rainy  season  tend  to  conserve  the  nitric  nitrogen  where 
fallow  soils  rapidly  lose  nitric  nitrogen  during  this  period. 

5.  The  rapidity  with  which  nitric  nitrogen  is  transformed  into 
protein  nitrogen  by  soil  microorganisms.     It  is  now  known  that 
there  are  within  the  soil  many  microorganisms  which  transform 
nitric  nitrogen  into  protein  nitrogen,  and  the  speed  with  which 
this  change  occurs  may  at  times  become  important;  work  at  the 
Utah  Experiment  Station  indicates  that  this  may  at  times  reach 
twenty  or  thirty  pounds  per  acre  yearly. 

The  factors  must  always  be  kept  in  mind  in  an  attempt  to  reach 
general  conclusions  from  any  special  cases,  yet  it  is  instructive  to 


LOSS  OF  NITRATES 


237 


examine  a  few  results  from  the  Rothamsted  Experiment  Station, 
as  compiled  by  Dr.  Hopkins: 


NITROGEN  IN  DRAINAGE   WATERS.      ROTHAMSTED  EXPERIMENTS 
AVERAGE   OF  12  YEARS   (OR  MORE). 


Month. 

Rainfall 
(inches). 

Bare  Soil  —  60-inch  Gauge. 

Wheat  Land. 

Drainage 
(inches). 

Nitrogen. 

Nitrogen. 

Per  Million 
of  Water. 

Pounds 
per  Acre. 

Per  Million 
of  Water. 

January  .... 
February 
March     .... 
April        .... 

2.13 
2.16 
1.70 
2.25 

1.93                8.9 
1.74                 9.1 
0.94                8.9 
0.79                9.0 

3.88 
3.57 
1.89 
1.61 

3.1 
4.0 
2.0 
1.9 

May  
June  
July   .      .      .      .      . 
August    .... 

2.48 
2.59 
2.85 
2.69 

0.79 
0.78 
0.62 
0.76 

9.1 
9.1 
11.8 
13.3 

1.63 
1.60 
1.66 
2.28 

0.9 
0.1 
0.1 
0.1 

September    . 
October  .... 
November    . 
December     . 

2.70 
3.12 
3.20 

2.34 

0.82 
1.68 
2.32 
1.88 

13.4 
11.9 
11.4 
10.6 

2.50 
4.53 
5.98 
4.51 

3.9 

4.6 
3.6 
4.8 

January-April    . 
May-  August 
Sept.-December 

8.24 
10.61 
11.36 

5.40 
2.95 
6.70 

9.0 
10.6 
11.8 

10.95 
7.17 
17.52 

2.8 
0.3 

4.2 

January-December 

30.21 

15.05 

10.5 

35.64 

2.4 

It  does  not  necessarily  follow  that  all  of  the  nitric  nitrogen 
which  is  carried  to  a  depth  of  sixty  inches  is  lost  to  the  growing 
plant,  for  in  work  at  the  Utah  Experiment  Station  the  author  and 
coworkers  have  found  in  the  spring  a  nitrate  belt  as  low  as  the 
seventh  and  eighth  foot-section.  These  nitrates  had  been  carried 
to  this  depth  by  the  winter  and  spring  water.  It  was  noted  that 
later  in  the  season  as  the  water  was  brought  to  the  surface  by 
capillarity  the  nitrates  also  returned,  and  by  June,  July  or  August, 
depending  upon  the  crop  grown  upon  the  soil  and  the  quantity  of 
irrigation  water  applied,  the  nitrate  belt  which  in  the  spring  was 
in  the  seventh  and  eighth  foot-section  had  reached  the  surface 
foot-section*.  Moreover,  the  deep-rooted  plants  of  the  arid  regions 
probably  feed  from  lower  depths  than  do  the  shallow-rooted  plants 
of  the  humid  regions. 

The  practical  conclusion  to  be  reached  from  these  results  is  that 
the  method  of  reducing  the  loss  by  leaching  is  by  growing  plants, 


238  NITRIFICATION 

the  roots  of  which  may  absorb  the  plant-food  as  rapidly  as  it  is  made 
available. 

The  loss  of  nitric  nitrogen  from  irrigated  soil  may  be  prevented 
by  the  judicious  use  of  irrigation  water.  Experiments  at  the  Utah 
Station  covering  a  period  of  fourteen  years  have  demonstrated  that 
the  application  of  fifteen  or  twenty  inches  of  irrigation  water, 
distributed  throughout  the  season,  to  deep  soil  causes  little,  if  any, 
loss  of  nitric  nitrogen  from  such  a  soil,  whereas  applications  of 'from 
twenty-five  to  thirty-seven  inches  similarly  distributed  causes  consid- 
erable diminution  in  the  crop  yield.  This  decrease  in  crop  yield  due 
to  excessive  quantities  of  water,  up  until  the  soil  becomes  water- 
logged, is  largely  due  to  the  rapid  washing  of  the  nitric  nitrogen 
beyond  the  feeding  area  of  the  plant  roots. 


REFERENCES. 

Lohnis:     "Handbuch  der  Landwirtschaftlichen  Bakteriologie." 

Lafar:     "Handbuch  der  Technischen  Mykologie,"  Dritter  Band. 

Kossowiez:     "Agriktdturmykologie,"  I — Bodenbakteriologie. 

Warington,  Robert:  "Six  Lectures  on  the  Investigations  at  the  Rothamsted 
Experimental  Station,"  U.  S.  Dept.  Agr.  Off.  Exp.  Sta.  Bui.  8. 

Voorhees,  Edw.  B.  and  Lipman,  Jacob  G.:  "A  Review  of  Investigations  in  Soil 
Bacteriology,"  U.  S.  Dept.  Agr.  Off.  Exp.  Sta.  Bui.  194. 

Chester,  Frederick  D.:  "Bacteria  of  the  Soil  in  Their  Relation  to  Agriculture." 
Penn.  Dept.  of  Agri.  Bui.  98. 

Greaves,  J.  E.,  Stewart,  R.,  and  Hirst,  C.  T.:  "Influence  of  Crop,  Season,  and 
Water  on  the  Bacterial  Activities  of  the  Soil."  Jour.  Agr.  Rsch.,  vol.  ix,  pp.  293-341. 

Gibbs,  W.  M.:  The  Isolation  and  Study  of  Nitrifying  Bacteria.  Soil  Science, 
1919,  vol.  viii,  pp.  427-481. 


CHAPTER  XXII. 
DENITRIFICATION. 

IT  has  been  known  for  a  long  time  that  under  conditions  which 
were  not  fully  understood,  there  may  and  often  does  result  a  loss 
of  soil  nitrogen.  Most  of  this  is  due  to  the  loss  of  nitrates  in  the 
drainage  water,  but  occasionally  there  are  losses  which  cannot  thus 
be  accounted  for.  This  has  been  attributed  to  various  causes, 
namely:  (1)  the  liberation  of  elementary  nitrogen  in  the  process 
of  decay  as  the  complex  protein  is  broken  down  into  simple  products, 
(2)  the  reduction  of  nitrates  or  nitrites  with  the  production  of 
ammonia  or  elementary  nitrogen,  (3)  the  transforming  of  nitrates 
and  ammonia  into  complex  proteins  through  the  action  of  micro- 
organisms. 

Often  the  losses  from  all  of  these  processes  have  been  grouped 
together  and  considered  as  denitrification.  This  vague  usage  of  the 
term  has  led  to  considerable  confusion  and  often  erroneous  con- 
clusions. But  the  term  denitrification  in  its  proper  and  more 
limited  sense  refers  only  to  the  complete  reduction  of  nitrates  with 
the  evolution  of  elementary  nitrogen.  It  is,  however,  often  applied 
in  a  broader  sense  to  include  all  deoxidation  processes  whereby 
nitrates  are  partly  or  wholly  reduced.  But,  as  pointed  out  by  Lip- 
man,  for  practical  agriculture  the  differences  are  of  some  moment. 
The  partial  reduction  of  nitrates  to  nitrites  or  to  ammonia  does  not 
necessarily  involve  a  loss  of  soil  nitrogen,  whereas  the  complete 
reduction  of  nitrates,  wherever  it  occurs,  must  of  necessity  involve 
such  losses.  Hence,  there  is  some  justification  for  referring  to  the 
partial  reduction  of  nitrates  as  denitrification.  But  it  is  not  justifi- 
able to  classify  under  the  head  of  denitrification  all  bacterial  activi- 
ties in  the  soil  which  lead  to  the  disappearance  of  nitrates  or  even 
to  the  diminution  in  the  total  store  of  soil  nitrogen.  For  it  has  been 
repeatedly  demonstrated  that  the  nitrates  may  completely  disappear 
without  involving  any  loss  of  nitrogen. 

Early  Theories.— We  have  seen  that  the  early  investigators 
attempted  to  explain  nitrification  by  purely  chemical  theories. 
This  was  also  true  with  denitrification.  Kuhlman,  as  early  as  1846, 
expressed  the  belief  that  nitric  nitrogen  may  be  reduced  in  the  soil 
to  ammonia  by  the  fermentation  of  organic  substances.  This  same 
idea  was  brought  out  twenty-one  years  later  by  both  Froehde  and 
Angus  Smith,  and  it  also  appears  prominently  in  the  writings  of 
Johnson  in  1870,  and  Davy  called  attention  to  the  fact  that  gaseous 
nitrogen  was  set  free  from  decomposing  organic  matter  in  the  soil, 


240  DENITRIFICATION 

The  splitting-off  of  free  nitrogen,  theoretically,  could  be  due  to  the 
reduction  of  nitrates  or  the  action  of  nitrous  acid  on  ammonia  or 
amins: 

2HNO3          =     2H2O      +     2O2       +       N2 
NHs  +     HNO2     =     N2         +     2H2O 

+     HNO2     =     N2         +       CH3OH     +     H2O 


Lawes,  Gilbert,  and  Pugh  showed  that  losses  of  nitrogen  often 
take  place  when  nitrogenous  organic  matter  was  made  into  an 
"agglutinated  condition"  with  water  and  allowed  to  decompose  in 
the  presence  of  air.  Practically  no  ammonia  could  be  detected. 
Three  possible  reactions  were  suggested  by  Lawes  and  Gilbert:  (1) 
an  oxidation  analogous  to  that  of  the  action  of  chlorin  on  ammonia 
by  which  free  nitrogen  is  evolved;  (2)  a  reduction  similar  to  that  of  a 
great  number  of  substances  upon  the  oxygen  compounds  of  nitrogen, 
by  which  the  oxygen  is  appropriated  and  the  nitrogen  set  free;  (3) 
the  two  actions  may  operate  in  succession,  the  one  to  the  other. 

Organisms  Concerned.—  Gay  on  and  Depetit,  however,  were  the 
first  to  announce  that  the  nitrogen  originated  from  the  nitrates. 
They  found  that  the  ferments  which  possess  this  power  need  organic 
matter  for  their  development  and  that  part  of  the  organic  nitrogen 
is  transformed  into  ammonia  and  perhaps  also  into  amido-deriva- 
tives  of  organic  substance. 

In  1886  they  isolated  two  organisms—  B.  denitrificans  ,  a  and  (3 
—capable  of  reducing  nitrates  with  the  evolution  of  gaseous  nitrogen. 
They  also  encountered  a  number  of  bacteria  that  could  reduce 
nitrates  to  nitrites,  and  since  that  time  the  denitrifiers  have  been 
found  very  widely  distributed. 

The  discovery  by  Breal  that  many  substances  of  organic  origin, 
and  especially  straw,  are  the  carriers  of  denitrifying  organisms  was 
of  far-reaching  importance.  These  organisms  are,  therefore,  carried 
with  the  litter  to  the  manure  and  later  with  the  manure  to  the  soil. 
It  was  found  by  Kunnemann  that  horse  manure  as  a  rule  contains 
denitrifying  organisms  and  these  are  usually  of  two  species,  one  of 
them  also  being  found  on  straw.  The  organism  found  only  in 
manure  reduces  nitrates  in  symbiosis  with  B.  coli  and  is  B.  denitrifi- 
cans I  of  Burri  and  Stutzer;  the  organism  occurring  in  both  manure 
and  straw  is  the  B.  denitrificans  II  of  the  same  authors.  The  denitri- 
fying organisms  are  less  frequently  present  in  cultivated  soil  and 
are  usually  a  different  kind.  Yet  they  are  abundant  in  the  upper 
layers  of  the  soil.  Bazarewski  found  them  irregularly  distributed 
in  the  deeper  layers  of  the  soil,  but  frequently  they  occurred  abun- 
dantly at  a  depth  of  one  meter.  They  have  also  been  found  to  a 
great  depth  in  the  Nebraska  soils.  Putnam  examined  201  species 
and  139  were  found  to  reduce  nitrates  to  nitrites,  while  the  other 
species  did  not  effect  this  reduction.  Burri  and  Stutzer  called  atten- 
tion to  the  fact  that  while  there  are  many  bacteria  which  will  reduce 


REACTION  OF  THE  MEtilA  241 

nitrates  to  nitrites,  those  capable  of  reducing  nitrates  to  ammonia 
or  of  setting  nitrogen  free  are  not  very  numerous.  Severin  isolated 
32  different  organisms  from  horse  manure  and  studied  29  of  these. 
Of  this  number  eight  species  were  capable  of  complete  reduction  of 
nitrates,  provided  the  nitrate  concentration  be  not  too  great.  Nine 
of  the  other  species  were  able  to  reduce  nitrates  to  nitrites. 

Stoklasa  divides  the  denitrifying  organisms  present  in  soils  and 
manures  into  two  principal  groups.  The  first  group  contains 
Clostridium  gelatinosum,  Proteus  vulgaris,  P.  zenkeri,  B.  ramosus 
n.  liquefaciens,  B.  mycoides,  B.  megatherium,  B.  subtilis,  and  B. 
prodigiosus,  and  others.  The  characteristic  of  these  organisms  is 
that  they  reduce  nitrates  to  ammonia  without  the  formation  of 
elementary  nitrogen.  The  second  group  contains  Bac.  hartlebi, 
B.  fluorescens  liquefaciens,  B.  pyocyaneum,  B.  stutzeri,  B.  filefaciens, 
B.  nitrovorum,  B.  centropunctatum,  B.  denitrificans ,  B.  coli  communis, 
B.  typhi-abdominalis,  and  others.  These  organisms  as  a  rule  reduce 
nitrates  to  elementary  nitrogen. 

Beer  yeasts  (Laurent),  especially  those  of  Duclaux,  reduce 
nitrates  at  20°  C.  Penicillum  glaucum,  mucor  racemosus,  and  similar 
organisms  also  have  a  reducing  power. 

It  is,  therefore,  true  that  whereas  active  nitrogen  fixation  is  a 
characteristic  possessed  by  only  a  limited  number  of  microorganisms, 
the  opposite— denitrification— appears  to  be  a  characteristic  pos- 
sessed by  many  widely  dissimilar  organisms. 

Reaction  of  the  Media.— The  denitrifying  organisms  are  similar 
to  the  nitrogen-fixing  organisms  in  that  they  require  a  slightly 
alkaline  medium  in  which  to  function.  Von  Caron  considers  that 
with  a  sugar  concentration  of  more  than  1  or  2  per  cent.,  a  depres- 
sion of  denitrification  occurs.  This  probably  is  due  to  the  formation 
of  fatty  acids  by  the  butyric  acid  ferments  of  the  soil.  When  it 
first  became  known  that  denitrification  may  take  place  in  manure 
heaps,  the  practice  became  prevalent  to  add  to  the  manure  sulphuric 
acid  to  prevent  denitrification.  It  was  found  that  sulphuric  acid  is 
extremely  active  in  preventing  denitrification  and  0.17  per  cent,  in 
the  cultural  medium  was  sufficient  to  prevent  the  development  of 
the  denitrifying  organisms. 

Ampola  and  Garino  found  that  the  addition  of  ground  peat 
showing  an  acidity  of  9.85  per  cent,  checked  the  activity  of  the 
denitrifying  organisms  as  well  as  that  of  other  ferments.  The 
organisms,  however,  were  not  killed  and  commenced  their  activity 
again  as  soon  as  the  acidity  was  neutralized.  The  soil  conditions 
are  favorable  to  the  neutralization  of  the  acid  of  the  peat,  and  thus 
the  restraining  effect  of  the  latter  on  the  denitrifying  organisms  is 
nullified.  Moreover,  an  acid  condition  which  would  restrain  deni- 
trifiers  in  soil  retards  the  other  beneficial  bacteria  and  higher  plants. 
Hence,  while  acids  may  be  used  at  times  with  some  success  on 
manures,  it  is  not  necessary  nor  practical  to  add  it  to  soils. 
16 


242  DENITRIFICATION 

An  excessive  alkaline  reaction  is  also  inimical  to  the  growth  and 
activity  of  denitrifying  organisms,  as  was  early  shown  by  Pfeiffer, 
but  the  application  of  such  large  quantities  of  caustic  lime  to  soil 
as  he  found  necessary  to  check  denitrification  tends  to  "burn  out" 
the  nitrogenous  organic  matter,  as  has  been  amply  demonstrated 
by  the  work  in  Pennsylvania. 

Food  Requirements.— The  food  requirements  of  the  denitrifiers 
are  quite  similar  to  those  of  other  soil  organisms.  They  can,  accord- 
ing to  Richards  and  Rolfo,  survive  in  purely  mineral  media,  but  in 
such  media  the  reduction  of  nitrates  takes  place  very  slowly  and 
incompletely.  Jensen  found  a  certain  relationship  between  the 
nitrate  destroyed  and  the  carbon  compounds  used.  No  denitrifica- 
tion takes  place  without  a  source  of  carbon.  The  optional  relation 
between  the  carbon  and  the  nitrate  used  was  found  by  von  Caron 
to  be  for  two  strong  denitrifiers—  B.  pyocyaneus  and  B.  fluorescens 
liquefaciens,  a  1  per  cent,  dextrose  to  1.6  per  cent,  potassium  nitrate. 
Reduction  of  the  nitrate  supply  far  below  that  of  carbon  greatly 
reduces  the  intensity  of  the  process.  Furthermore,  the  destructive 
fermentation  of  nitrates  depends  to  a  great  extent  on  the  character 
of  the  organic  substances  in  the  nutritive  medium,  some  being 
much  better  adapted  than  others  to  furnish  the  necessary  energy 
for  the  breaking  down  of  the  nitrates.  Stoklasa  claims  that  most 
of  the  denitrifying  bacteria  causes  no  reduction  of  nitrates  in  media 
where  chemically  pure  d.  levulose  and  d.  galactose  are  present.  Nor 
is  denitrification  favored  by  glucose  in  nutritive  solutions  (Stutzer) , 
but  is  promoted  by  the  presence  of  salts  of  organic  acids,  like  potas- 
sium lactate,  or  sodium  citrate.  The  reason  for  this  is  that  glucose 
is  not  as  suitable  for  furnishing  the  molecular  energy  required  for 
the  breaking  down  of  the  nitrates  as  are  the  salts  of  organic  acids. 
Stutzer  tried  four  different  organisms  and  found  that  they  possessed 
the  power  of  denitrification  in  a  different  degree.  Their  action  on 
the  different  meat  extracts  on  the  market  is  also  variable.  B. 
hartlebii  was  the  only  organism  tested  by  him  which  could  destroy 
nitrates  in  a  medium  containing  Liebig's  beef  extract.  He  suggested 
that  this  phenomenon  may  be  due  either  to  a  difference  in  chemical 
constitution  of  the  compounds  or  a  difference  in  ionization.  The 
knowledge  we  now  possess  concerning  the  specificity  of  enzymes 
would  lead  us  to  believe  the  former  to  be  the  true  explanation. 

Certain  of  the  most  widely  distributed  carbohydrates  in  soil  and 
manures,  as  for  example  xylose  and  arabinose,  are  not  especially 
good  nutrients  for  denitrifying  bacteria,  according  to  Stoklasa  and 
Vitek.  The  quantitative  relationship  which  they  found  to  occur  is 
widely  different  with  the  various  carbohydrates.  Of  the  bacteria 
which  reduce  nitrates  to  nitrites  and  finally  to  ammonia,  B.  mycoides 
reduced  20.69  per  cent,  of  the  nitrate  nitrogen  present  to  ammonia 
in  the  presence  of  glucose  1.9  per  cent,  in  the  presence  of  levulose, 
1.72  per  cent,  in  the  presence  of  galactose,  and  1.91  per  cent,  in  the 


METABOLISM.  OF  DENITRIFYING  ORGANISMS          243 

presence  of.  arabinose;  B.  subtilis,  2.41  in  the  presence  of  glucose, 
6.55  per  cent,  in  the  presence  of  levulose,  and  6.22  per  cent,  in  the 
presence  of  galactose;  Clostridium  gelatinosin,  45.55  per  cent,  in  the 
presence  of  arabinose,  and  9.68  per  cent,  in  the  presence  of  xylose; 
and  B.  prcdigiosus,  2.58  per  cent,  in  the  presence  of  xylose.  The 
reaction  was  in  all  cases  relatively  slow  and  was  not  alike  for  all  the 
sugars. 

Of  the  organisms  which  reduced  nitrates  to  free  nitrogen,  B', 
hartlebii  set  free  93.97  per  cent,  of  the  nitric  nitrogen  in  the  presence 
of  glucose,  87.59  per  cent,  in  the  presence  of  levulose,  84.66  per  cent, 
in  the  presence  of  galactose,  66.38  per  cent,  in  the  presence  of  arab- 
inose, 83.38  per  cent,  in  the  presence  of  xylose,  84.48  per  cent, 
in  the  presence  of  sucrose,  and  77.15  per  cent,  in  the  presence  of 
lactose;  B.  centropunctatum,  5.17  per  cent,  in  the  presence  of  glucose. 
B.  flitrowrum,  5.17  per  cent,  in  the  presence  of  levulose;  B.  coli 
communis,  5.34  per  cent,  in  the  presence  of  galactose;  and  Bad. 
fluorescens  liquefaciens ,  7.08  per  cent,  in  the  presence  of  arabinose. 
The  reaction  was  as  a  rule  very  intense  both  with  the  sugars  and 
with  the  salts  of  organic  acids,  especially  of  lactic  acid,  and  was 
accomplished  by  a  gradual  breaking  up  into  carbon  dioxid  and 
hydrogen  or  into  carbon  dioxid  and  water.  The  hydrogen  produced 
was  thought  to  play  a  very  important  reducing  role. 

Xylan  and  araban,  the  most  abundant  and  widely  distributed 
carbohydrate  materials  in  soils  and  manures,  yields  on  hydrolysis 
xylose  and  arabinose  which  are  very  poor  sources  ot  carbon  and 
energy  for  denitrifying  organisms.  However,  Stoklasa  and  Vitek 
found  that  the  typical  denitrifying  organism,  B.  hartlebii,  assimilated 
33.6  per  cent,  of  the  total  nitrate  nitrogen  in  a  nutritive  solution 
containing  arabinose  and  converted  it  into  protein  compounds. 

Sodium  citrate,  sodium  acetate  or  glycerin  added  to  a  soil  greatly 
increase  denitrification,  and  it  is  generally  considered  that  the  addi- 
tion of  starch,  straw,  rape  cake,  compost,  etc.,  to  a  soil  favors  deni- 
trification, whereas  well-rotted  manures,  rape  cake,  and  composts 
are  much  less  apt  to  have  this  effect. 

Metabolism  of  Denitrifying  Organisms.— Deherain  found  that 
reduction  was  more  rapid  in  closed  flasks  than  in  the  open  air,  the 
nitrogen  escaping  mainly  in  the  form  of  protoxid.  From  this  he 
argued  that  the  organisms,  being  deprived  of  the  necessary  oxygen 
of  the  air,  were  forced  to  appropriate  that  contained  in  the  nitrates 
and  thus  accomplish  their  reduction,  but  we  now  know  that  the 
denitrifiers  do  not  necessarily  require  anaerobic  conditions  for  deni- 
trification, but  do  require  a  readily  oxidizable  carbohydrate.  More- 
over, as  pointed  out  by  Stoklasa,  there  are  two  classes  of  denitrifiers 
—one  which  reduces  nitrates  to  elementary  nitrogen,  the  other 
which  reduces  it  only  to  ammonia.  Probably  in  both  groups  of 
organisms  the  first  steps  in  the  process  are  the  same.  The  carbo- 
hydrates are  broken  down  under  the  influence  of  the  microorganism 


244  DEN  I  TRIP  1C  A  TION 

into  lactic  acid,  alcohol,  and  carbon  dioxid.  The  nitrate  is  reduced 
to  nitrous  acid  and  this  in  turn  is  reduced  to  ammonia  or  element- 
ary nitrogen.  The  oxygen  so  obtained  is  utilized  by  the  microorgan- 
isms for  the  further  oxidation  of  the  carbohydrates,  and  it  is  in  this 
manner  that  the  organism  obtains  its  requisite  energy. 

Stoklasa  and  Vitek  believe  that  nitrous  acid  is  always  the  inter- 
mediate product  in  the  reduction  of  nitrates.  They  consider  that 
carbon  dioxid  and  hydrogen  are  produced  from  the  carbohydrates 
or  organic  acids  of  the  cultural  media  and  the  nascent  hydrogen 
combines  with  the  oxygen  of  the  nitrates  to  form  water  and  thus 
reduces  the  latter  to  nitrites.  Gayon  and  Depetit  give  this  formula: 

SCeHnOe     +     24KNO3     =     24KHCO3     +     6CO2     +     18H2O     +     12N2 

The  process  is  probably  due  to  enzymes.  Fred  was  able  to  demon- 
strate the  production  of  both  oxidases  and  peroxidases  by  B. 
denitrificans.  Hulme  considered  that  reduction  may  be  divided  into 
two  parts:  the  bacterial  reduction  and  the  enzymatic  reduction. 
However,  we  are  led  to  doubt  whether  either  is  due  to  a  true  enzyme, 
for  the  enzymes  which  have  been  obtained  in  impure  forms  are  not 
affected  by  heat  and  the  reducing  substances  are  not  specific,  as  is 
the  case  with  most  enzymes,  for  they  reduce  chlorates  to  chlorids, 
arsenates  to  arsenites,  and  ferricyanids  to  ferrocyanids  in  the  same 
manner  as  nitrates  are  reduced  to  nitrites. 

Influence  of  Water.— Many  of  the  results  obtained  on  denitrifica- 
tion  were  with  the  use  of  liquids,  and  it  is  now  known  that  denitrifica- 
tion  in  soils  progresses  differently  from  that  in  liquids,  depending 
upon  the  nature  of  the  bacteria  and  the  physical  conditions  of  the 
medium  in  which  they  are  situated.  In  liquids  and  very  wet  soils 
from  which  oxygen  is  excluded^  the  bacteria  take  their  oxygen  from 
the  nitrates  present  in  the  soil  and  thus  liberate  nitrogen,  but  in 
well  aerated  soils  this  does  not  occur,  as  the  bacteria  can  use  the 
oxygen  of  the  air. 

The  author  failed  to  find  any  evidence  of  denitrification  in  a  highly 
calcareous  soil  to  which  had  been  added  from  0  to  25  tons  per  acre 
of  manure  and  from  12.5  to  22.5  per  cent,  of  moisture,  as  may  be 
seen  from  the  following: 

Nitric  Gain  in 

nitrogen  total  nitrogen 

Treatment.  (per  cent.)  (per  cent.). 

12.5  per  cent,  of  water1 100  100 

15.0         "                               118  108 

17.5         "                               121  102 

20.0                                         121  104 

22.5         "                               123  108 

The  results  may  vary  with  different  soils,  but  Lemmermann  found 
that  in  three  greatly  dissimilar  soils  it  was  greatest  when  the  soil 
was  saturated. 

Therefore,   when  the  moisture  exceeds  certain  limits,  it  may 

1  The  soil  containing  12.5  per  cent,  of  water  was  taken  as  producing  100  per  cent. 


LOSSES  OF  NITRATES  FROM  MANURE  AND  SOIL      245 

promote  denitrification.  'Variations  in  the  moisture  within  the 
usual  limits,  however,  have  little  influence  upon  the  process. 

Temperature.— Kruger  considers  that  the  factors  which  exert  the 
greatest  influence  upon  denitrification  are  temperature  and  the 
mechanical  condition  of  the  material  which  furnishes  the  food  for 
the  organisms.  These  organisms  function  best  at  a  temperature 
which  is  high  enough  to  greatly  retard  nitrification.  They  act  very 
energetically  at  a  temperature  of  37°  C.  in  pure  cultures,  but  there  is 
some  evidence  (von  Bazarewski)"  that  in  mixed  cultures  they  func- 
tion better  at  a  lower  temperature.  These  factors  make  it  probable 
that  laboratory  results  on  denitrification  are  high  as  compared  with 
field  conditions  even  where  all  of  the  other  conditions  are  optimum. 

The  denitrifying  bacteria  are  more  resistant  to  light  and  drying 
than  are  the  nitrifying  or  nitrogen-fixing  organisms.  Ampola 
found  sunlight  to  have  no  effect  upon  two  denitrifiers  isolated  by 
him—  B.  denitrificans  V.  and  B.  denitrificans  VI.  In  pure  distilled 
water  these  organisms  were  capable  of  surviving  for  seven  months. 
When  dried,  B.  denitrificans  V  died  within  eight  weeks  and  B. 
denitrificans  VI  was  alive  and  active  at  the  end  of  five  months. 

Losses  of  Nitrates  from  Manure  and  Soil.— The  finding  of  the  deni- 
trifying bacteria  on  straw  and  in  manure,  together  with  the  estab- 
lishment of  the  fact  that  they  can  under  appropriate  conditions 
decompose  nitrates  with  the  evolution  of  gaseous  nitrogen,  led 
Wagner  in  1895  to  emphatically  declare  that  the  application  of  cow 
or  horse  manures  to  a  soil  is  often  not  only  unprofitable  but  harmful, 
that  when  applied  together  with  nitrates  they  cause,  by  virtue  of  the 
microorganisms  contained  in  them,  the  destruction  of  the  nitrates. 
More  than  that,  the  baneful  effects  do  not  stop  here,  for  the  nitrates 
as  they  are  gradually  formed  from  the  organic  matter  of  the  soil  are 
also  attacked  by  the  denitrifying  bacteria  and  their  nitrogen  set 
free.  In  reality,  then,  the  animal  manures  applied  are  not  only 
useless  in  themselves  but  are  harmful  because  of  their  destructive 
effects  on  the  oxidized  nitrogen  derived  from  other  sources. 

These  conclusions  were  criticized  by  Warington  who  pointed  out 
that  they  were  based  on  experiments  in  which  the  dressings  of  dung 
were  enormous  and  the  same  would  not  occur,  under  ordinary  prac- 
tice. The  next  year  a  serious  attempt  was  made  to  solve  the  problem. 
When  the  German  Agricultural  Association  called  for  a  united  effort 
on  the  part  of  the  German  experiment  stations,  offering  to  place  the 
necessary  means  at  their  disposal,  the  Experiment  Stations  of  Augs- 
burg, Darmstadt,  Jena,  Rostock,  Bonn,  and  Gottingen  responded. 
The  questions  to  be  answered  were  as  follows : 

1.  How  are  the  great  losses  of  nitrogen  that  take  place  in  the  decay 
of  organic  substances  to  be  explained?    How  much  of  the  nitrogen 
is  liberated  in  the  elementary  state  and  how  much  as  ammonia? 

2.  What  means  do  we  possess  of  checking  these  losses,  and  how 
does  the  substance  thus  employed  act? 


246  DENlTRfFlCATION 

The  published  reports  of  the  various  stations  are  voluminous  and 
only  the  general  conclusions  reached  can  be  considered  here.  They 
were  as  follows: 

1.  The  losses  of  ammonia  from  manure  are  comparatively  slight, 
but  the  setting  free  of  elementary  nitrogen  which  is  due  to  micro- 
organisms and  not  chemical  means  may  be  considerable. 

2.  With  a  limited  supply  of  air  in  manure,  the  loss  of  elementary 
nitrogen  and  of  organic  substance  are  not  extensive,  but  the  greater 
the  access  of  air  the  greater  the  loss  of  nitrogen,  in  some  cases 
becoming  as  great  as  40  or  50  per  cent. 

3.  In  ordinary  conservation  materials  when  applied  in  the  usual 
quantities,  do  not  stop  entirely  the  loss  of  nitrogen,  but  burnt  lime 
is  quite  effective  in  stopping  denitrification.    Solid  excreta  and  straw 
lose  their  nitrogen  very  slowly  and  no  conservation  material  is 
needed.    It  is  only  the  nitrogen  of  urine  which  requires  conservation. 

It  is  sometimes  found  that  the  addition  of  large  quantities  of 
organic  matter  to  a  soil  cause  a  decrease  in  crop  yield.  This  is 
especially  true  with  regard  to  the  carbohydrates  and  it  has  often 
been  interpreted  as  indicating  rapid  denitrification,  but  Pfeiffer 
and  Lemmermann  have  pointed  out  that  there  are  at  least  three 
factors  which  may  play  a  part,  namely:  (1)  direct  injury  to  the 
growing  plants  by  large  quantities  of  organic  matter;  (2)  fixation 
of  soluble  nitrogen  by  the  increased  activity  of  different  organisms; 
(3)  denitrification  proper. 

It  is  quite  probable  that  the  last  is  of  the  least  importance,  for 
Voorhees  and  Lipman  after  ten  years'  investigations  under  care- 
fully controlled  conditions  conclude  "  that  at  least  with  cow  manure, 
used  at  the  rate  of  sixteen  tons  per  annum  for  a  period  of  ten  years, 
no  destruction  of  nitrogen  takes  place.  In  view  of  the  long  duration 
of  the  experiment  and  of  the  comparatively  large  amounts  of 
manure  used  in  the  course  of  the  ten  seasons,  we  must  assume  that 
denitrification  is  not  a  phenomenon  of  economic  importance  in 
general  farming  and  under  average  field  conditions.  We  have  no 
hesitation  in  emphasizing  again  the  view  expressed  above— that 
under  the  wide  range  of  field  conditions,  denitrification  is  not  a 
phenomenon  of  economic  significance  to  the  general  farmer." 

Moreover,  at  Rothamsted  a  plot  of  ground,  0.001  acre  in  extent, 
has  been  kept  free  from  vegetation  by  hoeing  for  thirty-five  years. 
During  this  time  it  has  lost  one-third  of  its  original  stock  of  nitrogen, 
but  all  except  110  pounds  of  this  is  accounted  for  by  the  nitrates 
in  the  drainage  water,  as  may  be  seen  from  the  following : 


Nitrogen  in  soil. 
1870                1905 

Pounds  nitrogen 
per  acre. 
1870      1905    Loss    in 
35  years. 

Nitrogen  recovered 
as  nitrates. 
1870-1895 

Nitrogen  unaccounted 
for. 

0.146         0.102 

3500     2450     1050 

940 

110 

FUNCTION  OF  DENITRIFIERS 


247 


Russell,  commenting  upon  the  results,  states:  "The  experiment 
is  not  fine  enough  to  justify  any  discussion  of  the  missing  110  pounds, 
but  it  shows  that  the  loss  of  nitrogen  is  mainly  due  to  leaching  out 
of  nitrates." 

It  is  even  doubtful  if  denitrification  goes  on  to  any  appreciable 
extent  in  a  well  aerated  soil  even  though  it  contains  considerable 
nitrates.  The  nitrates,  however,  may  disappear  as  seen  from  the 
following  results  in  which  the  author  mixed  2  grams  of  dried  blood 
and  3724.8  parts  per  million  of  various  nitrates  with  100  grams  of 
soil  made  the  moisture  up  to  18  per  cent,  and  after  twenty-one 
days'  incubation  at  30°  C.  recovered  the  various  percentages  of 
nitric  nitrogen.  The  untreated  soil  was  taken  as  100  per  cent. 


NO,  added 
(p.  p.m.) 

Per  cent,  of  nitria  nitrogen  found  in  the  presence  of 

NaNOa 

KNOa 

Ca(N03)2. 

Mg(NO3)2 

Mn(N03)2 

Fe(NO3)2 

lone 

100 

100 

100 

100 

100 

100 

724.8    .      . 

-13.9 

-41.2 

-20.2 

-354.7 

-17.8 

7.8 

This  indicates  a  loss  of  nitrates  where  sodium,  potassium,  calcium, 
magnesium,  and  manganese  nitrates  had  been  applied  to  the  soil. 

An  analysis  of  the  soil  for  total  nitrogen  showed  a  loss  only  where 
the  potassium  nitrate  was  applied  to  the  soil,  and  in  this  case  it 
was  only  5.96  mgms.  in  place  of  41.2  per  cent.,  as  was  indicated  by 
the  first  results. 


Dri< 

jdblo 

rreatment. 
3d       + 

+ 
+ 
+ 
+ 
+ 
+ 

no 
84 

84 
84 
84 
84 
84 

nitrate 
.06  mg. 
.  06  mg. 
.  06  mg. 
.  06  mg. 
.  06  mg. 
.  06  mg. 

Gain  (  +)  or  loss  (  —  )  in 
nitrogen  over  soil  receiving 
no  nitrate. 

0.00 

N.N. 
N.N. 
N.N. 
N.N. 
N.N. 
N.N. 

as  KNO3 
as  NaNO3      . 
as  Mg  (NO3)2 
as  Fe(NO3)3  . 
as  Ca(NO3)2 
as  Mn(NO3)2 

.      .      .     -5.96mgs. 
...        1.34 
.      .      .      25.14 
.      .      .     37.04 
.      .      .      42.54 
.      .      .      48.54 

Function  of  Denitrifiers.— Huge  quantities  of  organic  and  inorganic 
nitrogen  find  their  way  into  the  septic  tanks  of  large  cities,  and 
much  of  this  is  returned  to  the  atmosphere  by  these  bacteria.  More- 
over, that  which  reaches  the  lakes  and  oceans  is  also  acted  upon  by 
denitrifying  bacteria;  hence,  they  play  a  part,  although  of  minor 
importance  in  the  nitrogen  cycle. 


REFERENCE. 

Voorhees,  Edward  B.,  and  Lipman,  Jacob  G.:  "A  Review  of  Investigations  in 
Soil  Bacteriology."  U.  S.  Dept.  Agr.  Off.  Exp.  Sta.  Bui.  194.  New  Jersey  Exp.  Sta. 
Ann.  Rpts.  1901  and  1902. 


CHAPTER  XXIII. 
AZOFICATION. 

THE  maintenance  of  the  nitrogen  supply  of  the  soil  is  the  phase 
of  soil  fertility  which  has  received  greatest  consideration  both  from 
the  scientist  and  from  the  practical  agriculturist.  Nitrogen  is  one 
of  the  more  expensive  commercial  fertilizers  and  is,  in  the  majority 
of  soils,  the  limiting  factor  of  crop  production.  The  supply  of  com- 
bined nitrogen  on  the  earth  is  comparatively  small  and  it  is  possible 
to  calculate  approximately  the  time  necessary  for  its  exhaustion. 
Basing  his  conclusion  on  such  a  calculation,  at  least  one  scientist 
has  predicted  dire  calamity  to  the  human  race  were  science  not  able 
soon  to  solve  this  problem.  Science  has  measured  up  to  its  require- 
ments in  this  regard,  for  the  synthetic  production  of  combined 
nitrogen  has  been  accomplished,  and  this  in  a  manner  so  highly 
satisfactory  that  it  is  able  to  compete  successfully  with  the  product 
of  natural  deposits.  Advancements  have  also  been  made  in  our 
knowledge  of  the  underlying  principles  influencing  the  natural  proc- 
esses which  govern  the  fixation  of  nitrogen  in  the  soil.  Although 
there  is  much  yet  to  be  learned  in  this  field  it  is  upon  the  control 
of  these  natural  processes  that  ultimate  success  will  be  based. 

Historical.— It  has  been  known  for  generations  that  uncropped  soils 
increase  in  fertility.  Less  ancient,  however,  is  the  knowledge  that 
this  increase  may  be  due  to  a  gain  of  nitrogen  in  the  abandoned 
soils.  Even  more  recent  than  this  is  the  knowledge  that  it  may  be 
due  to  bacteriological  action. 

In  the  middle  of  the  nineteenth  century  Boussingault  wrote: 
"Vegetable  earth  contains  living  organisms— germs— the  vitality 
of  which  is  suspended  by  drying  and  reestablished  under  favorable 
conditions  as  to  moisture  and  temperature."  He  also  hinted  at  the 
fact  that  these  microorganisms  take  part  in  the  process  of  nitrogen 
fixation.  He  spread  out  thinly  120  gm.  of  soil  in  a  shallow  glass  dish 
and  for  three  months  moistened  it  daily  with  water  free  from 
nitrogen  compounds.  At  the  end  of  this  time  analysis  showed  that 
it  had  lost  carbon,  but  had  gained  nitrogen.  It  was  not  until  thirty 
years  later  that  Hellriegel  and  Wilfarth  made  their  discovery  of 
nitrogen-fixation  by  symbiotic  organisms.  At  that  time  the  labora- 
tory technic  of  modern  bacteriology  was  still  undeveloped.  Since 
then,  however,  we  have  learned  much  concerning  the  relationship 
of  plants  to  free  and  combined  nitrogen  of  the  air  and  of  the  soil. 


HISTORICAL  249 

We  know  that  soil  gains  in*  nitrogen  are  often  due  to  microorganisms, 
either  living  free  in  the  soil  or  in  company  with  the  higher  plants. 
The  production  of  nitrogen  compounds  out  of  atmospheric  nitrogen 
by  bacteria  independent  of  higher  plants  is  designated  non-symbiotic 
nitrogen-fixation,  or  azofication.  When  fixation  is  accomplished  by 
bacteria,  living  in  connection  with  and  receiving  benefit  from  higher 
plants,  it  is  called  symbiotic  nitrogen-fixation. 

As  early  as  1883  Berthelot  undertook  the  study  of  soils  as 
regards  their  relationship  to  free  and  combined  nitrogen,  and  as  a 
result  of  these  studies  he  was  the  first  definitely  to  recognize  that 
gains  which  occur  in  bare  unsterilized  soils  are  due  to  microscopic 
organisms.  He  found  that  when  50  kgm.  of  arable  soil  were  exposed 
to  air  arid  to  rain  in  a  vessel  for  seven  months,  after  allowing  for  the 
small  amount  of  combined  nitrogen  brought  down  by  the  rain,  there 
was  a  gain  in  nitrogen  of  more  than  25  per  cent.  In  another  experi- 
ment in  which  the  soil  was  first  washed  free  from  nitrates,  there  was 
a  gain  of  46  per  cent.  Many  other  experiments  showed  gains  from 
10  to  15  per  cent.  Berthelot  was  not  content  with  the  bare  knowl- 
edge that  nitrogen  is  fixed  in  the  soil  by  living  organisms,  but  con- 
tinued his  work  with  the  idea  of  isolating  some  of  these  organisms. 
With  the  aid  of  Guignard,  he  made  soil  inoculation  into  sterile 
bouillon  and  from  this  prepared  gelatin  plates.  Cultures  were  taken 
from  the  colonies  growing  on  the  plates  and  bacteria  were  tested 
for  their  nitrogen-fixing  power.  His  results  were  conclusive  that 
there  exist  within  the  soil  chlorophyll-free  bacteria  capable  of  fixing 
atmospheric  nitrogen.  His  work  had  shown  that  these  organisms 
act  best  at  summer  temperatures,  between  50°  and  104°  F.,  in  the 
presence  of  a  good  supply  of  oxygen,  a  proportion  of  water  in  the 
soil  not  exceeding  12  to  15  per  cent,  and  not  falling  below  2  to  3 
per  cent. 

They  require  carbon,  hydrogen  and  enough  combined  nitrogen 
to  promote  initial  growth.  The  nitrogen,  gained  by  the  soil  was 
proteinaceous  in  nature,  being  insoluble  in  water.  Although  some 
of  his  soils  had  gained  large  quantities  of  nitrogen,  he  considered 
that  the  fixation  of  atmospheric  nitrogen  by  microorganisms  has 
its  limits,  since  the  organisms  isolated  drew  from  the  atmosphere 
only  so  long  as  the  amount  fixed  in  the  medium  was  not  great.  Heat- 
ing the  soil  to  230°  F.  immediately  stopped  the  process. 

Prior  to  this  a  number  of  chemists,  notably  Konig  and  Kiesow, 
Armsby,  Birner,  Kellner,  Deherain  and  Avery  had  found  that  when 
organic  matter  in  one  form  or  another  undergoes  fermentation  there 
is  frequently  an  increase  of  nitrogen  in  the  fermenting  substance. 
Armsby  states  it  thus:  "We  must  conclude  that  decaying  organic 
substances  in  the  presence  of  caustic  alkali  are  able  to  fix  free 
nitrogen  without  the  gain  being  manifest  as  nitric  acid  or  ammonia, 
and  probably  without  the  formation  of  these  bodies."  His  explana- 


250  AZOF  1C  AT  ION 

tion  of  the  process  was  that  the  nascent  hydrogen  evolved  during 
the  fermentation  process  reacted  with  the  free  nitrogen  of  the  air. 
Others  considered  that  the  active  agents  were  compounds  of  iron, 
manganese,  and  lime  existing  in  the  soil  and  in  some  way  acting 
as  catalytic  agents. 

Berthelot's  discovery  interested  Winogradsky  who  commenced 
work  which  eventually  bridged  the  chasm.  He  employed,  as  a 
medium,  a  nutritive  solution  free  from  combined  nitrogen,  but  con- 
taining mineral  salts  and  dextrose.  Fifteen  separate  species  of  soil 
bacteria  were  isolated,  but  only  one— a  long  sporebearing  bacillus 
which  developed  normally  in  the  absence  of  combined  nitrogen  and 
seemed  to  produce  butyric  fermentation— fixed  nitrogen  to  any 
appreciable  degree.  Quantitative  tests  showed  that  the  maximum 
fixation  was  attained  where  no  combined  nitrogen  was  purposel 
added,  and  that  on  the  addition  of  such,  fixation  of  nitrogen  was 
diminished.  For  example,  several  determinations  gave  the  following 
results: 

N  as  NH3  in  dextrose  solution    .      .      2.1         4.2         6.4         8.5         21.2 
Nfixed 7.0         5.0         5.5         3.6  2.2 

The  presence  of  combined  nitrogen  tends  to  decrease  fixation. 
He  concluded  that  in  order  for  any  gain  to  be  made,  the  ratio  of  the 
combined  nitrogen  to  the  sugar  should  not  exceed  6:1000.  Because 
of  the  characteristic  formation  of  clostridia  in  his  cultures,  Wino- 
gradsky named  the  organism  Clostridium  pasteurianum.  The  con- 
clusion which  the  author  reached,  however,  was  that  the  power  of 
fixing  nitrogen  is  not  general  among  microorganisms,  but  confined 
to  a  few  special  forms. 

Following  Winogradsky,  Caron  made  some  interesting  discoveries. 
He  found  that  soils  under  leafy  crops  contain  greater  numbers  of 
bacteria  than  those  under  grasses.  He  also  observed  that  the  bac- 
terial flora  of  soils  in  the  spring  are  different  from  *those  in  the  fall 
both  quantitatively  and  qualitatively.  He  used  in  vegetation 
experiments  pure  cultures  of  the  bacteria  most  frequently 
encountered  in  natural  soils.  Some  soils  were  inoculated  with 
bouillon  culture,  whereas  others  received  only  sterile  bouillon. 
The  crop  yields  were  usually  in  favor  of  the  inoculated  plots,  but 
showed  variations  from  season  to  season.  Exceptionally  good  results 
were  obtained  with  a  sporebearing  bacillus  which  he  termed  Bacillm 
ellenbachensis. 

Caron's  work  led  to  the  commercial  exploitation  of  his  cultures, 
one  of  which,  "alinit,"  was  the  subject  of  much  study  and  contro- 
versy. This  culture  was  found  to  contain,  according  to  Severin,  two 
closely-related  bacilli  which  he  chose  to  designate  as  B.  ellenbachensis 
A  and  B.  These  had  the  power  to  fix  nitrogen  to  some  extent.  Tests 


HISTORICAL  251 

with  "alinit,"  however,  have  not  confirmed  to  any  great  extent  the 
claims  of  its  exploiters. 

In  1901  Beijerinck's  investigations  led  to  an  extremely  important 
addition  to  the  history  of  non-symbiotic  nitrogen-fixation.  He 
described  a  new  group  of  large  aerobic  bacilli  to  which  he  gave  the 
generic  name  Azotobacter. 

,  In  an  early  paper  published  by  Beijerinck  and  van  Delden,  they 
maintain  that  Azotobacter  are  incapable  of  fixing  appreciable 
quantities  of  nitrogen  in  pure  culture,  but  are  dependent  to  a  large 
extent  on  Granulobacter ,  Radiobacter,  Aerobacter.  They  considered 
that  in  mixed  cultures  the  Granulobacter,  Radiobacter,  and  Aerobacter 
possess  the  power  of  fixing  nitrogen  in  the  presence  of  Azotobacter, 
which  grows  at  the  expense  of  the  combined  nitrogen  escaping  from 
them  into  the  solution. 

A  little  later  Gerlach  and  Vogel  succeeded  in  isolating  from  soil 
the  Azotobacter  of  Beijerinck  and  in  showing  that  in  pure  cultures 
and  in  the  presence  of  salts  of  organic  acids,  Azotobacter  are  capable 
of  active  nitrogen-fixation.  They  obtained  a  fixation  of  9  mgm. 
of  nitrogen  in  a  1  per  cent,  solution  of  grape  sugar.  But  Beijerinck 
challenged  this  assertion,  claiming  that  their  cultures  were  not  pure 
but  were  mixed  with  other  forms  difficult  to  separate.  The  claims 
of  Gerlach  and  Vogel  were  substantiated  by  the  work  of  Freuden- 
reich,  Koch  and  Lipman.  The  latter  not  only  showed  that  the 
Azotobacter  possess  the  power  of  fixing  nitrogen  in  pure  cultures, 
but  he  explained  the  failures  recorded  by  others. 

Although  not  necessary,  the  presence  of  other  organisms  often 
proves  advantageous..  Lipman  found  that  in  the  presence  of  such 
forms  as  B.  radiobacter  and  B.  levaniformus  the  nitrogen-fixation  is 
faster  and  goes  on  at  a  more  regular  rate. 

To  the  two  species  of  Azotobacter— A.  chroococcum  and  A.  agilis— 
described  by  Beijerinck  and  van  Delden,  Lipman,  added  A.  mne- 
landii,  A.  beijerinckii,  and  A.  woodstownii.  Later  Lohnis  and 
Westermann  described  A.  vitreum,  and  after  a  study  of  21  cultures 
of  various  Azotobacter  concluded  that  they  represented  only  four 
types.  A.  chroococcum  is  most  widely  distributed  in  the  soils  so  far 
studied.  ^^^ 

The  discussion  of  the  subject  thus  far  has  been  more  or  less  con- 
fined to  the  Azotobacter,  but  investigations  of  Beijerinck  and  van 
Delden,  Lohnis,  Moore,  Chester,  Bredemann  and  others  have 
brought  to  light  other  microorganisms  having  the  power  to  fix 
nitrogen.  Among  these  are  B.  mesentericus  (which  fixes  appreciable 
quantities  of  nitrogen),  B.  pneumonia,  B.  lactis  viscosus,  B.  radio- 
bacter,  B.  prodigiosus,  B.  asterosporus  and  B.  amylobacter. 

Bredemann,  after  a  careful  study  of  the  morphological  and  physio- 
logical characteristics  of  eleven  "original  species"  of  other  investi- 
gators and  of  sixteen  cultures  prepared  by  himself  from  various  soils, 
concluded  that  all  belong  to  the  single  species  B.  amylobacter  of 


252  AZOFICATION     . 

van  Tieghem.  Since  this,  however,  there  has  been  described  at 
least  one  aerobic  clostridium.  Moreover,  Omelianski  considers 
that  the  Clostridium  pasteurianum,  isolated  from  the  Russian  soils, 
is  clearly  a  morphologically  distinct  race.  An  idea  of  the  activity 
of  some  organisms  in  fixing  nitrogen  may  be  obtained  from  the  fol- 
lowing results  reported  by  Lohnis.  In  every  100  c.c.  of  1  per  cent, 
mannite,  or  grape  sugar  soil  extract,  there  was  fixed,  in  the  course 
of  three  weeks,  nitrogen  as  follows: 

Mg. 

Bact.  chrysogloea  .      .    ' 1.4 

Bact.  tartaricus 0.3 

Bact.  lipsiense 0.2 

C.  B.  Lipman  tested  18  organisms,  including  yeasts,  pseudo- 
yeasts,  and  molds,  nearly  all  of  which  showed  a  more  or  less  pro- 
nounced power  of  fixing  atmospheric  nitrogen. 

Pringsheim  has  isolated  from  ordinary  garden  soil  certain  thermo- 
philic  organisms  which  fix  from  3  to  6  mgm.  of  nitrogen  per  gram  of 
dextrose  when  incubated  at  61°  C.  in  a  Winogradsky's  solution  to 
which  a  little  soil  extract  was  added.  Duggar  and  Davis  have 
recently  investigated  the  subject  of  the  fixation  of  nitrogen  by  the 
filamentous  fungi,  Aspergillus  niger,  Macrosporium  commune,  Peni- 
cillium  digitatum,  Pexpansum,  Glomerella,  Gossypii,  and  Phoma 
beta,  and  of  these  only  the  last-named  was  definitely  proved  to  be 
able  to  fix  nitrogen.  It  is  thus  seen  that  the  power  of  fixing  nitrogen 
is  a  characteristic  possessed  by  many  microorganisms,  in  contradic- 
tion to  the  supposition  of  Winogradsky  that  this^power  is  limited 
to  a  particular,  or,  at  most,  a  few  species.  This  is  especially  empha- 
sized by  the  recent  work  of  Emerson  who  examined  soil  which 
contained  2,400,000  organisms  per  gram  which  would  develop  on 
nitrogen-free  media.  Of  these,  97  per  cent,  possessed  the  power 
of  fixing  nitrogen;  they  constituted  at  least  four  distinct  groups. 
Nevertheless,  the  most  important  group  yet  discovered  is  the 
Azotobacter,  and  it  is  with  these  mainly  that  this  chapter  deals. 

Distribution. —The  nitrogen-fixing  organisms  are  widely  distributed, 
occurring  in  most  soils.  Lipman  and  Burgess,  who  studied  the  nitro- 
gen-fixing flora,  especially  those  of  the  Azotobacter  group,  of  46  soils 
from  Egypt,  India,  Japan,  China,  Syria,  the  Hawaiian  Islands, 
Guatemala,  Costa  Rica,  Spain,  Italy,  Russia,  Mexico,  Asia  Minor, 
Canada,  Unalaska,  Samoa,  Australia,  Tahiti,  Belgium,  Queensland, 
and  the  Galapagos  Islands,  found  every  soil  possessed  the  power  of 
fixing  nitrogen  in  mannite  solution.  About  one-third  of  the  soils 
contained  Azotobacter',  frequently  the  same  soil  showed  the  presence 
of  two  or  three  different  species  of  Azotobacter.  A.  chroococcum, 
however,  was  the  most  prominent.  It  was  also  found  most  widely 
distributed  in  the  various  soils.  Groenewege  found  Azotobacter  in  all 
but  one  of  a  series  of  Java  soils. 

Several  hundred  Utah  soils  have  been  examined  and  all  found  to 
fix  nitrogen,  many  of  them  without  the  addition  of  carbohydrates. 


DISTRIBUTION  253 

Aerobic  Azotobacter  are  present  in  nearly  all  Utah  soils.  Hutchinson 
found  the  Azotobacter  in  all  the  Indian  soils  examined.  They  occur 
in  cultivated  more  frequently  and  in  greater  numbers  than  in  virgin 
soils.  This  probably  accounts  for  the  much  higher  nitrogen-fixing 
power  of  cultivated  soils. 

Azotobacter  were  found  in  only  two  out  of  64  localities  in  the  soils 
of  Danish  forests.  Both  of  the  soils  which  gave  positive  tests  were 
from  beechwood  forests  and  contained  calcium  carbonate.  Although 
the  soils  of  these  forests  rarely  contain  enough  carbonate  to  effervesce 
they  are  usually  neutral  or  slightly  alkaline.  They  contain  calcium, 
but  in  forms  other  than  the  carbonate.  It  is  generally  understood 
that  Azotobacter  occur  commonly  in  soils  which  contain  sufficient 
calcium  carbonate  to  effervesce  when  acid  is  added  and  that  they 
scarcely  ever  occur  in  acid  soils.  Their  disappearance  from  soil  is 
usually  due  to  the  absence  of  basic  substances,  especially  of  calcium 
and  magnesium  carbonate,  and  not  to  the  presence  of  toxic  sub- 
stances. However,  they  are  frequently  not  present  in  peaty  soils, 
where  their  absence  cannot  be  attributed  to  a  lack  of  lime. 

The  aerobic  nitrogen-fixers  are  probably  more  widely  distributed 
in  soils  than  are  the  anaerobic,  for,  although  both  groups  are  gener- 
ally found  in  the  Russian  soils,  the  aerobic  are  found  in  the  sands 
of  Kirghese  steppes  and  in  the  peat  soils  of  the  Province  of  Arch- 
angel in  which  the  anaerobic  forms  are  absent.  Anaerobic  nitrogen- 
fixers  are,  however,  quite  widely  distributed  in  soils  and  are  at  times 
found  on  the  leaves  of  forests  trees. 

The  nitrogen-fixing  organisms  are  confined  almost  entirely  to  the 
first  three  feet  of  soil,  although  they  have  been  found  in  soil  at  all 
depths  down  to  the  tenth  foot  in  .the  very  favorably  constituted 
loose  soils  of  Nebraska. 

They  are  most  active  in  the  upper  few  inches  of  soil,  as  is  indicated 
by  results  obtained  by  Ashby. 

Average 

Depth  nitrogen  fixed. 

Soil.  inches.  mgm. 

Little  Hoos 10  9.23 

Little  Hoos 20  7.29 

Little  Hoos 30  4.60 

Reports  on  some  Hawaiian  soils  show  them  to  be  equally  active 
at  all  depths  to  4  feet,  but  this  must  be  considered  an  exception, 
for  the  examination  of  numerous  soils  in  Utah  has  shown  a  gradual 
decrease  in  nitrogen-fixing  powers  with  depth.  The  average  of 
several  hundred  determinations,  in  both  solution  and  soil  media, 
are  given  below : 

Nitrogen  fixed   in 

Nitrogen  fixed  in  100  cc.  of  Ashby's 

100  gm.  of  soil  +  solution  with  1.5 

1.5  gm.  of  mannite.  gm.  of  mannite. 

Depth  of  sample.  mgm.  mgm. 

Firstfoot 5.28  2.11 

Second  foot 2.42  0.77 

Third  foot       .,...,,.      1.55  0.58 


254  AZOFICATION 

These  samples  were  collected  with  such  great  care  that  there  was 
no  possibility  of  the  mixing  of  one  foot  section  with  another.  It  is 
interesting  to  note  that  while  the  actual  gain  in  nitrogen  per  gram 
of  mannite  is  over  twice  as  great  in  the  soil  as  in  the  solution,  vet 
the  relative  gain  per  foot  section  is  the  same  in  both.  There  is 
about  one-half  as  much  nitrogen  fixed  in  the  second  as  in  the  first 
foot,  and  one-fourth  as  much  in  the  third  as  in  the  first. 

The  nitrogen-fixing  organisms  are  not  confined  to  the  soil  alone, 
for  Beijerinck  and  van  Delden  first  isolated  Azotobacter  agilis  from 
canal  water  in  Holland.  Azotobacter  chroococcum  and  B.  Clostridium 
pasteurianum  are  both  found  in  many  fresh  and  salt  waters,  living 
on  algse  and  plankton  organism. 

Reaction  of  the  Media.— The  distribution  and  the  physiological 
efficiency  of  the  nitrogen-fixing  organisms,  especially  of  the  Azoto- 
bacter species,  are  governed  by  the  physical  and  chemical  properties 
of  the  soil,  foremost  among  which  is  the  basicity  of  the  soil,  namely, 
its  calcium  or  magnesium  carbonate  content.^  Ashby  bases  his 
method  for  obtaining  pure  cultures  of  Azotobacter  upon  this  property, 
for  he  finds  that  by  picking  out  the  crystals  of  the  carbonate  from 
the  soil  and  seeding  them  into  nitrogen-free  media  the  likelihood  of 
obtaining  the  organism  is  greatly  increased.  The  addition  of  calcium 
carbonate  to  a  soil  often  increases  its  azofying  power,  the  extent  of 
which  increase  depends  on  the  lime  requirements  of  the  soil  and  on 
the  fineness  of  the  added  limestone. 

Christensen  has  suggested  that  the  Azotobacter  be  used  as  an  index 
to  the  lime  requirements  of  a  soil.  The  test  should  include  both  a 
search  for  the  organism  in  the  soil  and  a  test  of  their  ability  to  grow 
when  inoculated  into  the  soil.  He  and  Larson  examined  more  than 
one  hundred  soils  of  known  lime  requirement.  They  determined 
the  carbon  dioxid  set  free  by  acids,  the  amount  of  calcium  dissolved 
by  an  ammonium  chlorid  solution,  the  behavior  of  the  soil  toward 
litmus,  and  the  biological  test.  The  result  of  this  test  was  that  the 
biological  test  agreed  with  the  known  condition  in  90  per  cent,  of  the 
cases,  the  ammonium  chlorid  in  50  per  cent.,  the  litmus  in  40  per 
cent.,  and  the  carbon  dioxid  failed  more  often  than  not  to  indicate 
the  correct  condition  of  the  soil. 

Fischer  failed  to  find  Azotobacter  in  a  heavy  loam  soil  containing 
only  0.145  per  cent,  of  lime,  while  adjoining  limed  plots  had  an 
Azotobacter  flora.  The  quantity  of  calcium  carbonate  which  must 
be  added  to  obtain  maximum  fixation  varies  with  the  soil. 

A  West  Virginia  Dekalb  silt  loam,  which  required  0.175  per  cent. 
of  calcium  carbonate  to  render  it  neutral  by  the  Veitch  method, 
gave  greatest  nitrogen  fixation  when  0.375  per  cent,  of  calcium 
carbonate  was  added.  Above  this  concentration  azofication 
decreased,  but  when  phosphorus  was  applied  with  the  lime  it  was  not 
toxic  even  when  present  in  quantities  as  great  as  0.5  per  cent.  It  is 


REACTION  OF  MEDIA  255 

certain  that  large  quantities  of  calcium  carbonate  may  be  present 
in  soil  without  injury  to  the  azofiers. 

The  author  found  numerous  Azotobacter  and  a  very  active  nitro- 
gen-fixation in  a  soil  43  per  cent,  of  which  was  calcium  and  mag- 
nesium carbonate. 

The  organisms  develop  normally  in  the  presence  of  either  calcium 
or  magnesium  carbonate,  but  in  liquid  cultures  the  film  develops 
earlier  and  it  contains  less  foreign  organism  in  the  presence  of  mag- 
nesium carbonate  than  in  the  presence  of  calcium  carbonate.  The 
actual  nitrogen  fixed,  as  reported  by  Ashby,  is  also  greater  where 
the  magnesium  carbonate  is  used.  This  he  attributes  to  the  sup- 
pression by  the  magnesium  of  foreign  organisms,  especially  of  the 
butyric  acid  ferments. 

There  is,  however,  a  marked  difference  in  the  action  of  calcium 
carbonate  and  magnesium  carbonate  when  they  are  applied  in  large 
quantities.  Lipman  and  Burgess  found  the  calcium  carbonate  stimu- 
lating and  never  toxic  to  Azotobacter  chroococcum  in  concentrations 
up  to  2  per  cent,  in  mannite  solution.  The  magnesium  carbonate 
was  sharply  toxic  in  higher  concentrations  up  to  2  per  cent,  in 
mannite  solution.  The  magnesium  carbonate  was  sharply  toxic 
in  higher  concentrations  above  0.1  to  0.2  per  cent,  in  such  cultures. 
The  calcium  salt  is  without  effect  when  added  to  most  soils  up  to 
1.4  per  cent.,  but  the  magnesium  carbonate  is  even  more  toxic  in 
soils  than  in  solutions.  Moreover,  their  work  indicates  that  calcium 
exerts  a  protective  influence,  in  both  soils  and  solutions,  against 
the  toxic  influence  of  magnesium.  The  best  ratio  of  calcium  to 
magnesium  varies  with  solution  and  soil. 

In  many  soils  lime  increases  the  nitrogen  fixed,  for  Krzemieniewski 
found  limed  soil  to  fix  in  ten  days  17.52  mgm.  of  nitrogen,  whereas 
adjoining  unlimed  soil  fixed  only  7.15  mgm.  There  is,  however,  the 
possibility  of  applying  too  large  a  quantity  of  the  caustic  lime  and 
thereby  decreasing  nitrogen-fixation,  a  condition  which  has  never 
been  experienced  in  the  use  of  the  carbonate. 

Von  Feilitzen,  however,  found  neither  a  direct  relationship  be- 
tween lime  content  of  moor  soil  and  the  development  of  Azotobacter, 
nor  relationship  between  their  development  and  the  reaction  of  the 
soil.  But  this  only  serves  to  illustrate  the  fact  that/although  lime 
and  neutral  or  slightly  alkaline  media  are  essential,  they  will  not" 
ensure  a  rich  Azotobacter  flora  in  a  soil  unless  all  other  conditions 
are  optimum.  1  Remy  found  sodium  and  potassium  carbonate  less 
favorable  for  nitrogen-fixation  than  was  calcium  or  magnesium. 

So  far  as  the  writer  is  aware,  Krainsky  is  the  only  worker  who  has 
found  sodium  carbonate  more  favorable  than  calcium  carbonate. 
This  may  have  been  due  to  the  sodium  carbonate's  liberating  plant- 
food  which  was  in  the  soil  in  an  insoluble  form  but  which  was  essen- 
tial to  the  development  of  Azotobacter,  Mockeridge  has  found  that 


256 


AZOFICATION 


the  presence  of  sodium  salts  is  unnecessary  and  depressing  at  least 
to  the  growth  of  Azotobacter.  The  beneficial  effect  ascribed  to  sodium 
chlorid  solution  in  inoculating  agar  plates  is  due  to  the  fact  that 
this  liquid  is  isotonic  with  the  cell  content  solution,  but  the  sodium 
hydroxid  is  a  far  less  advantageous  neutralizing  agent  than  is  cal- 
cium or  magnesium  carbonate.  Furthermore,  Lipman  failed  to 
stimulate  the  azofiers  with  any  of  the  sodium  salts. 

Food  Requirements  of  the  Azofiers.'— These  organisms  probably 
require  for  their  nutrition  the  same  elements  as  do  the  higher  plants, 
namely  carbon,  hydrogen,  oxygen,  nitrogen,  potassium,  phosphorus, 
sulphur,  calcium,  magnesium,  and  iron,  and  possibly  aluminum  and 
manganese. 

They  obtain  their  carbon  and  hydrogen  from  organic  compounds, 
preferably  from  carbohydrates,  which  are  considered  in  detail  under 
sources  of  energy.  Oxygen  is  obtained  either  from  the  atmosphere 
or  from  combined  sources  depending  on  the  species  and  the  condi- 
tions under  which  they  are  grown. 

A  marked  difference  between  these  and  the  higher  plants  is  that 
they  possess  the  power  of  obtaining  their  nitrogen  from  the  air,  but 
in  the  presence  of  combined  nitrogen  they  obtain  but  little  from  the 
a'lr.  '  Lipman,  Stranak,  Heinze,  and  Stoklasa  found  that  small 
quantities  of  nitrates  stimulated  Azotobacter,  whereas  large  quanti- 
ties discouraged  nitrogen-fixation  since  the  organisms  live  on  the 
nitrates.  This  is  the  case  whether  the  nitrates  are  added  to  the  soil 
or  to  the  solution  in  which  nitrogen-fixation  is  taking  place.  Cole- 
man  considers  this  action  as  due  to  several  different  factors :  namely, 
(a)  a  direct  toxic  action  of  the  salt,  (6)  antagonism  of  other  organ- 
isms which  it  favors,  (c)  the  using  up  of  the  energy  supply  by 
these  organisms,  and  (d)  the  discouragement  of  fixation  by  the  use 
of  sodium  nitrate.AThe  last  would  seem  to  be  the  most  important 
factor  when  viewed  in  connection  with  the  following  results  reported 
by  Hills: 


Relative  per  cent,  of  nitrogen  fixed. 

-  j     ,        .                         ,                f 

ivCl&tivc  number  01  organisms. 

Sterilized  soil. 

Unsterilized  soil. 

^Treatment 

nitrate. 

Mgm. 

KNO3 

NaNOj 

Ca(NOa)j 

NaNOj 

Ca(N03)2 

NaNOs 

Ca(N03)2 

0 

100 

100 

100 

100 

100 

100 

100 

10 

348 

191 

362 

100 

105 

240 

219 

50 

8210 

3150 

4528 

342 

371 

500 

444 

150 

12 

117 

763 

200 

0 

0 

0 

352 

467 

879 

557 

FOOD  REQUIREMENTS  OF  THE  AZOFIERS  257 

The  number  of  organisms  developing  and  the  nitrogen  fixed  in 
the  one  receiving  no  nitrate  is  taken  as  100  per  cent. 

It  is  quite  evident  from  these  results  that  although  nitrates  cause 
more  active  multiplication  of  Azotobacter,  it  greatly  reduces  their 
physiological  efficiency.  The  organisms  used  by  Hills  had  probably 
grown  for  a  long  time  on  media  poor  in  nitrogen,  and  their  ability 
to  fix  nitrogen  was,  therefore,  high.  But  would  they  continue  to 
exert  this  power  if  grown  on  media  rich  in  nitrogen?  The  evidence 
points  strongly  to  the  conclusion  that  they  would  not.  It  is  certain, 
however,  that  the  nitrates  are  toxic  in  comparatively  low  concentra- 
tions. Nitrates  and  ammonium  sulphate  are  rather  effective  in  stimu- 
lating nitrogen-fixation  when  the  Azotobacter  are  grown  in  connection 
with  the  cellulose  ferments.  Even  here,  however,  large  quantities 
decrease  this  power.  In  pure  cultures  ammonium  sulphate  seriously 
retards  nitrogen-fixation,  whereas  the  nitrogen  of  humus,  even  in 
large  quantities,  appears  to  have  no  serious  retarding  influence. 
Nevertheless,  a  high  nitrogen  content  of  soils  seems  to  be  unfavor- 
able to  vigorous  nitrogen-fixation. 

Whether  this  would  be  the  case  where  the  nitrate  content  of  the 
soils  is  kept  low  but  with  the  readily  decomposable  protein  nitrogen 
high,  is  yet  to  be  answered.  Hiltner  and  Stormer  consider  that  when 
the  nitrogen  content  of  the  soil  passes  beyond  a  certain  limit,  the 
decay  bacteria  increase  rapidly,  and  in  the  struggle  for  existence 
they  are  able,  with  the  advantage  at  their  disposal,  to  suppress  the 
more  slowly  growing  Azotobacter. 

Potassium  is  essential  to  the  higher  plants  and  cannot  be  replaced 
entirely  by  related  elements,  yet  Gerlach  and  Vogel  early  reached 
the  conclusion  that  potassium  and  magnesium  are  not  essential  to 
the  Azotobacter.  Their  results  were,  however,  generally  considered 
erroneous,  for  while  as  much  nitrogen  was  fixed  in  twenty  days 
without  as  with  potassium,  after  forty  days  there  was  no  further 
fixation  in  the  solution  without  potassium,  but  in  its  presence  the 
nitrogen  gain  nearly  doubled.  It  was,  therefore,  argued  that  the 
traces  of  potassium  left  in  the  chemicals  and  dissolved  from  the 
glass  during  sterilization  had  been  enough  to  permit  development 
for  a  time.  If  these  elements  are  essential,  it  must  be  in  extremely 
minute  quantities,  for  Vogel,  using  the  purest  chemicals  obtainable, 
was  able  to  prepare  potassium-free  media  in  which  the  Azotobacter 
developed.  He  did  find,  however,  that  potassium  favors  their 
development. 

Phosphorus  is  required  by  these  organisms,  large  quantities  being 
used  for  the  building  of  the  nucleo-proteins  and  phospho-proteins  in 
which  their  bodies  are  extremely  rich.  Moreover,  it  greatly  acceler- 
ates the  reaction  and  economizes  the  carbohydrates;  hence  it  is 
rather  evident  that  phosphorus  plays  a  very  essential  part  in  Azoto- 
bacter metabolism.  y  Possibly  in  the  early  stages  of  the  process  a 
17 


258  AZOFICATION 

definite  chemical  reaction  occurs  between  the  phosphate  and  the 
carbohydrate  similar  to  that  occurring  in  alcoholic  fermentation. 

I.  2C6Hi2O6     +     2R2HPO4    -»     2CO2     +     2H2O     +     C6HioO4(PO4R2)2     + 

2C2HeO 
II.  C6H10O4(PO4R2)2     +     2H2O    -*     C6H12O6     +     2R2HPO4 

The  Azotobacter  are  able  to  utilize  the  phosphorus  of  di-  and 
tri-basic  sodium  and  potassium  phosphate  and  of  dibasic  calcium 
phosphate. 

Mockeridge  obtained  an  increase  of  23  per  cent,  in  nitrogen  fixa- 
tion with  basic  slag.  There  were  two  maxima,  one  with  0.4  per  cent, 
the  other  with  1.0  per  cent.  slag.  This  is  attributed  to  the  stimulat- 
ing effect  of  the  iron  and  manganese  in  the  slag,  the  maximum  effect 
of  one  being  produced  at  0.4  per  cent.,  the  other  at  1.0  per  cent. 
The  tribasic  calcium  phosphate— bone  ash,  iron,  and  aluminum 
phosphate— all  serve  only  as  difficultly  available  sources  of  phos- 
phorus. Raw  rock  phosphate  and  bone  meal  fail  entirely  to  furnish 
enough  available  phosphorus  for  the  development  of  Azotobacter. 

The  addition  of  phosphorus  to  a  soil  often  greatly  increases  azofi- 
cation. 

Without  With 

phosphorus.  phosphorus. 

Treatment.  .  mgm.  mgm. 

No  lime ....0.6  0.9 

Lime .....1.5  4.6 

Moreover,  Christensen  has  found  soils  in  which  phosphorus  is  the 
limiting  element  in  Azotobacter  growth.  He  entertains  the  hope  that, 
in  view  of  the  relationship  between  Azotobacter  growth  and  lime  and 
phosphorus,  it  will  become  eventually  possible  by  the  determin- 
ation of  bacterial  food  requirements  to  secure  a  general  expression 
for  the  soil  content  of  plant-food  available  to  crops.  He  further 
suggests  that  where  a  mannitol  solution  free  from  phosphorus 
produces  a  vigorous  growth  of  Azotobacter  after  inoculation  with  a 
soil,  it  may  be  assumed  that  the  soil  is  not  deficient  in  available 
phosphorus.  Dzierzbicki  notes  that  if  soils  are  deficient  in  available 
lime,  phosphoric  acid,  or  potash,  nitrogen-fixing  bacteria,  such  as 
Azotobacter,  are  either  entirely  absent  or  present  only  in  small 
numbers. 

There  is  a  definite  relationship  between  the  carbon  and  phos- 
phorus content  of  a  soil  and  the  nitrogen  assimilated.  According 
to  Stoklasa,  Azotobacter  assimilates  from  5.0  to  5.7  grams  of  free 
nitrogen  for  every  gram  of  phosphorus  used.  Although  these 
organisms  are  directly  dependent  upon  a  readily  available  supply 
of  phosphorus  to  promote  growth,  they  do  not  change  it  into  the 
organic  form  as  rapidly  as  do  the  ammonifying  bacteria. 

Sulphur  is  required  by  the  azofiers  possibly  for  the  formation  of  the 


ORGANIC  SOIL  CONSTITUENTS  259 

proteinaceous  material  of  their  bodies.  It  is  certain  that  the  benefit 
derived  by  Azotobacter  from  the  sulphates  of  iron  and  calcium  is  due 
in  a  large  measure  to  the  sulphur  which  these  compounds  supply.  No 
evidence  has  as  yet  been  produced  which  would  lead  us  to  believe 
that  the  organisms  can  use  sulphur  as  a  source  of  energy. 

Calcium  carbonate  and  calcium  oxid,  in  addition  to  furnishing  a 
base  which  neutralizes  the  acid  formed  in  the  metabolic  processes 
of  the  Azotobacter,  also  furnish  calcium  to  the  organism.  Christensen 
brought  out  the  fact  that  Azotobacter  can  derive  their  calcium  from 
dibasic  calcium  phosphate  and  some  calcium  salts  of  organic  acids. 
They  could  not,  however,  utilize  the  calcium  of  tribasic  phosphate, 
of  calcium  chlorid  or  sulphate. 

Iron  is  essential  and  either  the  ferric  or  ferrous  sulphate  is  especially 
beneficial.  Rosing  found  the  amount  of  nitrogen  fixed  increased 
from  2.23  mgms.  to  10.3  mgms.  per  gram  of  mannite  when  iron 
sulphate  was  added  to  the  cultural  media.  This  is  due,  in  a  great 
degree,  to  the  iron  which  serves  as  food  for  the  organisms,  yet  its 
colloidal  nature  may  play  a  part,  for  both  organic  and  inorganic 
colloidal  substances  have  an  especially  favorable  action  on  Azoto- 
bacter, although  the  action  of  the  inorganic  colloids  is  fully  manifest 
only  in  the  presence  of  organic  colloids.  If  used  alone,  large  quan- 
tities of  the  ferric  hydroxid  are  essential  for  the  maximum  effect, 
but  in  the  presence  of  organic  colloids  very  small  quantities  of  iron 
are  effective.  This  has  been  attributed  to  the  action  of  the  colloidal 
iron  which  adsorbs  the  nitrogen  and  oxygen  of  the  air  and  brings 
them  into  more  intimate  contact  with  the  Azotobacter.  This  would 
not  only  accelerate  the  normal  processes  of  the  aerobic  Azotobacter 
by  furnishing  theni  with  nitrogen  and  oxygen,  but  it  w^ould  tend  to 
suppress  the  anaerobic  processes  which  are  extremely  wasteful  of 
the  food.  According  to  Kaserer,  these  organisms  also  require 
aluminum.  Although  this  may  accelerate,  it  has  not  been  proved 
to  be  essential  to  their  growth. 

While  not  essential  to  the  organisms,  manganese  is  an  extremely 
active  catalyzer  in  increasing  proportions  up  to  6  mgm.  per  100  c.c. 
of  media.  Above  this  concentration  the  reaction  falls  off  rapidly, 
and  at  20  mgm.  it  is  less  than  in  the  absence  of  manganese.  It  is 
oxidized  by  Azotobacter,  and  in  the  proportion  of  1  part  to  200,000 
parts  of  soil  it  is  an  active  stimulant.  Olaru  considers  it  likely  that 
the  increased  yield  obtained  after  the  application  of  manganese 
compounds  to  a  soil  is  due  to  its  accelerating  the  action  of  the 
nitrogen-fixing  organisms  of  ^he  soil. 

Organic  Soil  Constituents.— Reed  found  urea,  glycocol,  formamid, 
and  allantoin  active  in  depressing  nitrogen-fixation*  This  he 
attributes  to  the  compounds  furnishing  the  Azotobacter  an  available 
source  of  combined  nitrogen  and  not  to  a  direct  toxicity*  But 
Walton  found  that  the  addition  of  urea,  peptone,  acetamid,  aspar- 


260  AZOFICATION 

agin,  and  casein  to  culture  media  had  only  a  slight  influence  on  the 
fixation  of  nitrogen  by  Azotobacter. 

Caffeine,  alloxan,  betain,  trimethylamin,  legumin,  cinnamic  acid, 
aspartic  acid,  asparagin,  hippuric  acid,  creatin,  creatinin,  xanthin, 
and  hypoxanthin,  are  all  toxic  to  Azotobacter  even  in  small  quantities. 
Only  the  first  two  have  been  tested  in  concentrations  dilute  enough 
to  stimulate,  which  is  remarkable,  as  many  of  these  compounds 
stimulate  the  higher  plants  and  some  can  be  utilized  directly  by 
the  plant. 

Esculin,  vanillin,  daphnetin,  cumarin,  pyrocatechin,  heliotropin, 
arbutin,  resorcin,  pyrogallol,  phloroglucin,  hydroquinon,  salicylic 
aldehyd,  oxalic  acid,  quinic  acid,  dihydrostearic  acid,  rhamnose 
and  borneol,  on  the  other  hand,  do  not  stimulate  in  any  concentra- 
tion. Nor  are  they  toxic  until  fairly  large  quantities  have  been 
added.  In  this  regard  the  nitrogen-fixing  organisms  appear  to  differ 
widely  from  the  nitrifying  bacteria  and  higher  plants.  The  resist- 
ance of  the  nitrogen-fixers  to  various  chemicals  has  likewise  been 
called  to  our  attention  by  Lipman  in  his  study  of  the  influence  of 
alkalies  on  nitrogen-fixation. 

Influence  of  Colloids.— It  was  recognized  early  in  the  study  of 
nitrogen-fixation  that  when  sterilized  soil  is  added  to  a  nutritive 
medium  it  greatly  increased  the  quantity  of  nitrogen  fixed/  This 
condition  is  due  to  several  factors  and  is  partly  explained  by 
Krzemieniewski's  results  wherein  he  found  that  nitrogen-fixation  is 
decidedly  increased  by  the  addition  of  soil  humus,  either  as  free 
humic  acid  or  as  salts  of  potassium,  sodium  or  calcium.  Kaserer 
maintains  that  this  is  due  to. the  inorganic  nutrients,  especially  to 
aluminum  and  silicic  acid  supplied  to  the  .microorganisms  through 
the  humus.  This  is  probably  true  in  part,  for  the  fixation  varies  with 
the  humus  derived  from  different  sources.  Moreover,  artificial 
humus,  prepared  by  boiling  sugar  with  acids,  fails  to  stimulate. 

That  much  of  the  beneficial  effect  is  due  to  the  constituents  in  the 
humus  appears  likely  from  the  results  obtained  by  Sohngen  who 
found  that  colloidal  iron  oxid,  aluminum  oxid,  and  silicon  oxid  all 
greatly  stimulated  the  nitrogen-fixing  powers  of  Azotobacter  chroo- 
coccum.  This  he  attributed  to  the  absorption  of  oxygen  and  nitrogen 
by  the  colloid,  which  he  maintains  would  make  them  more  readily 
available  to  the  organisms.  The  boiling  of  natural  humus  with 
hydrochloric  acid  would  either  remove  the  foreign  material  or  change 
it  from  the  colloidal  form,  and  thus;  as  has  been  found  to  be  the 
case,  render  it  inert.  Lohnis  and  Green  take  exception  to  this 
explanation,  for  they  found  no  adsorptive  action  exerted  by  humus 
on  either  the  nitrogen  or  the  oxygen.  Furthermore,  Rosing  found 
that  he  could  stimulate  just  as  effectively  with  iron  as  with  humic 
acids.  But  much  larger  quantities  of  colloidal  iron  are  required 
when  it  is  used  singly  than  when  used  in  conjunction  with  an  organic 


SOURCES  OF  ENERGY  FOR  THE  AZOTOBACTER         261 

colloid.  The  extent  of  the  stimulation  resulting  varies  with  the  form 
in  which  the  iron  is  applied  and  is  most  effective  in  the  form  of  the 
hydroxid  and  in  the  presence  of  cane  sugar.  In  this  case  it  is 
probable  that  the  saccharate  is  the  active  substance.  Hence,  the 
contradictory  results  reported  may  be  due  to  the  different  min- 
eral constituents  of  the  humus. 

These  facts  make  it  certain  that  Colloids  of  the  metals  act  as 
stimulants  to  nitrogen-fixing  bacteria,  as  does  also  crude  humus. 
Carefully  purified  humates  do  not  possess  this  property,  but  it  is 
possessed  by  the  aqueous  extract,  the  alcoholic  extract,  and  the 
phosphotungstic  fraction  of  the  aqueous  extract  from  "bacterized" 
peatv  Whether  this  influence  is  due  to  a  catalytic  effect,  as  suggested 
by  Sohngen,  or  whether  the  substance  furnished  a  direct  source  of 
nutritive  material  is  not  clear  at  the  present  time. 

Moreover,  the  colloid  may  act  as  a  protection  to  the  organism 
against  poison;  for,  when  10  parts  per  million  of  soluble  arsenic  is 
maintained  in  a  soil,  it  acts  as  a  stimulant  to  Azotobacter.  If,  how- 
ever, this  proportion  is  added  to  the  Ashby  nutritive  solution  it  stops 
all  nitrogen-fixation.  This  is  due  in  part  to  the  adsorption  of  the 
arsenic  by  the  soil.  This  adsorption  would  have  to  be  attributed 
largely  to  the  silica  compounds,  for  the  nitrogen-fixing  organisms  are 
stimulated  by  arsenic  in  quartz  free  from  organic  colloids.  This 
could  readily  be  due  to  the  arsenic  becoming  concentrated  at  the 
surface  layers  of  the  silica,  leaving  the  inner  part  of  the  water  film 
comparatively  free  from  arsenic,  in  which  part  of  the  water  film  the 
microorganisms  multiply  and  carry  on  their  metabolic  processes. 
This  being  the  case,  one  should  and  probably  could  find  a  water 
solution  weak  enough  to  stimulate  bacteria.  A  great  difference, 
however,  between  the  solution  and  the  sand-culture  method  is  the 
greater  aeration  in  the  sand.  That  the  aeration  of  a  culture  medium 
does  play  an  important  part  in  determining  the  activity  of  the 
nitrogen-fixing  powers  of  a  soil  is  strikingly  brought  out  in  Fig.  18, 
page  124. 

Sources  of  Energy  for  the  Azotobacter.— The  nitrogen-fixing  organ- 
isms differ  widely  from  other  plants  in  their  energy  requirements. 
This  is  due  to  the  fact  that  they  are  carrying  on  en  do  thermic  reac- 
tions in  which  nitrogen  is  concerned.  This  necessitates  a  greater 
supply  of  energy  than  is  required  by  other  bacteria.  They  are 
similar  to  most  other  bacteria  in  that  this  energy  must  be  supplied 
by  an  organic  compound,  preferably  one  of  the  carbohydrates./ 

Berthelot  in  his  early  work  maintained  that  the  gains  in  nitrogen 
noted  in  some  soils  were  due  to  the  action  of  biological  agents  on 
the  humus  of  the  soil.  This  was  followed  by  the  observation  of 
others  that  when  forest  leaves  are  allowed  to  decompose  in  soil  there 
is  an  increase  in  its  nitrogen  content.  Koch  in  1907  increased 
nitrogen-fixation  by  the  addition  to  soil  of  dextrose,  cane  sugar  or 


262  AZOFICATION 

starch,  but  there  was  practically  no  increase  when  straw,  filter 
paper  or  buckwheat  was  applied.  Yet  Stoklasa  showed  that  the 
decomposition  products  of  these  substances  acted  as  a  valuable 
source  of  energy  to  the  Azotobacter,  and  Stranak  considered  that  the 
pentosans  of  the  soil  are  of  the  greatest  importance  in  the  assimila- 
tion of  nitrogen  by  soil  bacteria. 

A  fair  idea  of  the  great  variety  and  relative  efficiency  of  substances 
which  may  serve  as  a  source  of  energy  to  the  azofiers  may  be  obtained 
from  the  work  of  Lohnis  and  Pillai.  They  inoculated  a  nutritive 
solution  with  10  gm.  of  soil  and  after  ten  days  determined  the  gain 
in  nitrogen. 

Nitrogen  fixed 

Substance  added.  afterlO  days 

mgm. 

Mannite 9.40 

Xylose 9.54 

Lactose 9.12 

Levulose 8.52 

Inulin 7.72 

Galactose        7.86 

Maltose 7.44 

Arabinose        .      .      .      .• 7.62 

Dextrin 7.18 

Sucrose 8.60 

Dextrose 4.62 

Starch 3.36 

Sodium  tartrate 2.82 

Glycerin .  1.68 

Sodium  succinate       .      .      .      .      . 2.96 

Calcium  lactate   .      .      . 2.49 

Sodium  citrate .      .      .      .      .      .      .      .  1.42 

Sodium  propionate 1.10 

Potassium  oxalate .0.12 

Calcium  butyrate 0.02 

Humus .      .      .  -0.96 

Other  workers  have  noted  larger  gains  of  nitrogen  than  those 
noted  by  Lohnis  and  Pillai,  but  they  can  readily  be  attributed  to  the 
time  of  incubation— in  this  case,  ten  days  being  far  too  short  for  the 
complete  utilization  of  the  carbonaceous  substance  applied;  the 
species  of  nitrogen-fixers  which  are  bringing  about  the  change;  and 
whether  pure  or  mixed  cultures  are  used.  The  order  of  effectiveness 
noted  above,  however,  is  that  recognized  by  most  workers.  Brown 
and  Allison,  however,  do  report  results  in  which  greater  fixation 
was  obtained  with  dextrose  than  with  mannite.  But  in  this  case, 
calcium  or  sodium  carbonate  seems  to  be  even  more  necessary  than 
it  is  with  the  mannite.  Moreover,  some  species  utilize  one  carbo- 
hydrate most  effectively  and  another  species  a  different  one.  To 
this  list  may  be  added  malate,  gum  tragacanth,  ethylene  glycol, 
methyl,  ethyl,  and  propyl  alcohols,  lactic,  malic,  succinic  and  gly- 
collic  acids.  Fatty  acids  are  readily  utilized,  the  amount  of  nitrogen 
fixed  being  greater  with  the  increased  molecular  weight,  from  1.47 
mgm.  with  formic  acid,  to  6.08  mgm.  with  butyric  acid.  Most  of  the 


SOURCES  OF  ENERGY  FOR  THE  AZOTOBACTER         263 

naturally  occurring  glucosides  and  many  benzin  derivatives  are 
unsuitable  as  sources  of  energy  for  Azotobacter.  Molasses,  which 
should  serve  as  a  useful  source  of  energy,  often  results  in  a  loss  of 
nitrogen  when  applied  to  the  soil.  This  may  be  due  to  the  time  of 
applying,  for  Peck  maintains  that  molasses  applied  to  a  land  lying 
fallow  at  an  interval  of  several  weeks  before  planting  of  the  crop 
may  produce  beneficial  results  by  increasing  nitrogen-fixation. 

Beijerinck  early  recognized  that  certain  decomposition  products 
of  cellulose  can  also  serve  as  sources  of  energy  for  Azotobacter,  and 
Pringsheim  found  that  Clostridium  americanum  does  not  fix  atmos- 
pheric nitrogen  on  sterilized  cellulose  unless  other  carbohydrates 
like  dextrose,  lactose,  mannitol,  or  sucrose  are  present.  However, 
in  the  presence  of  cellulose,  Clostridium  will  fix  nitrogen  and  this 
more  efficiently  than  it  will  in  the  regular  carbohydrate  medium. 
The  same  holds  for  agar.  Just  how  completely  cellulose  must  be 
broken  down  before  it  can  be  utilized  by  Azotobacter  is  not  definitely 
known,  but  it  is  known  that  Azotobacter  cannot  utilize  cellobiose 
except  when  grown  in  conjunction  with  Aspergillus  niger~GT  other 
organisms.  It  is,  therefore,  certain  that  the  products  which  are 
utilized  by  the  Azotobacter  are  comparatively  simple. 

/Cellulose  when  applied  to  the  soil  may  serve  as  a  valuable  source 
of  energy,  provided  sufficient  time  is  allowed  for  its  decomposition. 
The  cellulose  ferment  is  probably  the  most  efficient  organism  in  the 
soil  in  bringing  about  this  decomposition.  But  the  number  of  soil 
fungi  which  possess  this  power  is  largex 

Hoppe  Seyler  thinks  that  cellulose  is  decomposed  according  to  the 
following  formula:  (a)  the  hydration  of  the  cellulose  with  the 
formation  of  hexose, 

C6Hi2O«. 


the  destruction  of  the  carbohydrate  with  the  formation  of  equal 
quantities  of  carbon  dioxid  and  methane. 


None  of  the  cellulose  ferments  studied  by  McBeth,  however,  yielded 
gaseous  products  when  acting  on  cellulose  or  sugar;  hence  the 
Azotobacter  probably  gets  from  the  cellulose  ferments,  pentoses 
and  hexoses,  and  similar  products  upon  which  they  can  readily  fix 
nitrogen. 

At  times  in  fermenting  straw  and  manure,  the  thermophilic 
anaerobic  bacteria  play  a  major  part,  in  which  case  fatty  acids 
probably  make  up  the  greater  part  of  the  end  products. 

It  is  claimed  by  Dvarak  that  substances  with  low  carbon  and  high 
oxygen  content  are  usually  the  best  sources  of  energy  for  A.  chroo- 
coccum,  which  assimilated  5.73  mgm.  of  free  nitrogen  per  100  gm. 
of  carbon  in  pine  leaves  as  compared  with  1237.9  mgm.  per  100  gm. 


264  AZOFICATION 

of  carbon  in  red  clover.     He  obtained  for  other  substances  the 
following  results: 

1456.5  mgm.  of  nitrogen  per  100  gm.  as  glucose. 
280.4  mgm.  of  nitrogen  per  100  gm.  as  cornstalks. 
596.8  mgm.  of  nitrogen  per  100  gm.  in  stalks  and  root  residues  of 
corn. 

325.4  mgm.  of  nitrogen  per  100  gm.  in  wheat  straw. 

The  carbon — nitrogen  ratio  in  compounds  is  no  indication  of  their 
value  to  nitrogen-fixing  organisms,  for  non-leguminous  hays  and 
straws  are  utilized  just  as  effectively  as  are  the  legumes.  Mockeridge 
found  that  the  ratios  of  nitrogen  fixed  to  the  heat  of  combustion  with 
the  four  lower  fatty  acids  is  almost  constant.  The  same  holds  true 
with  starch,  dextrin,  and  gum  arabic,  when  allowance  is  made  for 
experimental  error,  which  is  greater  with  these  compounds  than 
with  the  simpler  compounds.  This  close  relationship  is  not,  how- 
ever, graduated  and  no  such  uniformity  is  observed  with  the  series 
of  monohydric  alcohols. 

The  quantity  of  nitrogen  fixed  per  gram  of  carbohydrate  varies 
greatly  with  the  species.  Winogradsky  found  Clostridium  pasteuria- 
num  to  assimilate  2  to  3  mgms.  of  nitrogen  for  each  gram  of  sugar. 
But  this  like  other  anaerobic  organisms  is  very  wasteful  of  energy, 
leaving  much  of  it  in  the  butyric  acid,  acetic  acid,  and  butyl  alcohol 
formed.  In  the  experiments  of  Bredemann  with  B.  amylobacter  and 
of  Pringsheim  with  Clostridium  americanum,  the  amounts  fixed  were 
at  times  much  larger.  Much  greater  fixations  have  been  reported 
with  Azotobacter,  and  Lipman  has  obtained  as  high  as  15  to  20  mgms. 
of  nitrogen  per  gram  of  mannite  assimilated  by  A.  vinelandii.  This 
quantity  is  considerably  greater  than  that  fixed  by  any  of  the  other 
members  of  the  group. 

Koch  and  Seydel  claim  that  the  usual  method  of  estimating  the 
nitrogen-fixing  powers  of  Azotobacter  is  erroneous,  as  it  does  not 
represent  accurately  the  intensity  of  the  process.  In  a  series  of 
experiments  made  by  them,  the  amounts  of  nitrogen  fixed  per  gram 
of  dextrose  used  were  53,  70  to  80,  20  to  30,  and  5  to  8  mgms.  on  the 
first,  second,  third,  seventh,  and  eighth  days,  respectively. 

Krainsky  considers  that  there  should  be  sufficient  organic  matter 
in  the  soil  to  permit  that  for  1  part  of  nitrogen  formed  there  will  be 
90  parts  of  carbon  for  the  use  of  the  organism.  The  organisms,  how- 
ever, utilize  the  carbohydrates  more  economically  when  only  small 
quantities  are  present.  Walton  finds  with  Indian  soil  that  highest 
fixation  is  obtained  per  gram  of  mannite  when  10  grams  are  used  in 
1  liter  of  nutritive  solution.  Young,  vigorously  growing  cultures 
usually  fix  more  nitrogen  than  the  older  ones.  The  nitrogen  fixed 
is  greatest  in  the  first  stages  of  the  growth  of  the  organisms,  as  is 
seen  from  Fig.  33  from  the  work  of  Omelianski. 

vThe  efficiency  of  these  organisms  is,  therefore,  greatest  when  they 


SOURCES  OF  ENERGY  FOR  THE  AZOTOBACTER 


265 


are  rapidly  multiplying,  and  it  decreases  as  their  metabolic  products 
accumulate.  /  Hoffmann  and  Hammer  claim  this  to  be  due  in  impure 
cultures  to  a  loss  of  nitrogen  or  free  ammonia  occasioned  by  the 
decomposition  of  the  cells  of  Azotobacter.  This  explanation  would 
hardly  hold  in  the  presence  of  pure  cultures,  unless  we  ascribe  the 
breaking  down  to  an  autolytic  ferment  secreted  by  the  Azotobdcter 
cell.  According  to  Koch  and  Seydel  this  indicates  that  in  the  latter 
stages  of  fixation,  when  there  occurs  an  accumulation  of  nitrogenous 
material  in  the  medium,  the  organisms  employ  the  carbohydrates 


3.  5 


3.0 


2.5 


2.0 


J.5 


10 


0.5 


0  S  70  J5  20  25          30  35          H 

FIG.  33. — Graph  showing  the  fixation  of  nitrogen  and  decomposition  of  sugar  in 
mixed  cultures  of  Azotobacter  chrodcoccum  and  Clostridium  pasteurianum. 

for  other  purposes  than  for  nitrogen-fixation.  >  Under  natural  con- 
ditions in  the  soil  this  accumulation  and  concentration  of  nitrogenous 
material  by  the  Azotobacter  is  not  likely  to  occur;  hence,  they  assume 
that  the  organism  will  continue  fixing  nitrogen  at  the  high  ratio 
noted  in  the  early  part  of  laboratory  experiments .\ 

The  quantity  of  nitrogen  fixed,  however,  is  dependent  upon  factors 
other  than  the  source  of  energy;  e.  g.,  Krzemieniewski  found  with 
A.  chrodcoccum  that  the  addition  of  humates  to  the  cultural  solutions 
increased  the  nitrogen  fixed  from  a  maximum  of  2.4  mgm.  to  14.9 


266  AZOF1CATION 

mgm.  Moreover,  Krainsky  found  Azotobacter  to  utilize  from  100 
to  200  gm.  of  sugar  in  the  assimilation  of  1  gm.  nitrogen  when 
grown  in  solution,  but  when  grown  on  sand  it  required  only  11  to 
30  gm.  for  the  same  fixation. 

They  utilize  their  energy  more  economically  in  the  presence  of  a 
liberal  supply  of  phosphorus  than  when  the  quantity  of  available 
phosphorus  is  limited.  This  accounts,  in  a  measure,  for  the  high 
fixation  noted  in  most  Utah  soils. 

Manure.— It  has  been  known  for  a  long  time  that  humus  exerts 
a  highly  favorable  effect  on  nitrogen-fixation.  The  great  question, 
however,  has  been  as  to  the  manner  of  action.  Humus,  being  such 
a  complex,  variable  substance,  varies  greatly  in  action,  depending 
upon  its  source.  Remy  considered  that  some  of  the  products  from 
humus  are  favorable  sources  of  organic  matter  for  Azotobacter. 
Definite  and  valuable  information  is  furnished  by  the  work  of 
Lohnis  and  Green.  They  worked  with  mixed  cultures  of  A.  chroo- 
coccum,  A.  beijerinckii,  A.  vinelandii,  and  A.  mtrium  in  Beijerinck's 
mannite  solution  with  various  forms  of  organic  matter. 

Nitrogen  fixed  in 
100  cc.  of  solution 

Material.  after  3  weeks. 

mgm. 

Fresh  straw    . 10.0 

Fresh  stable  manure 9.8 

Fresh  peat      .      .      .      .      .      .      .      .      .      .      . "   .      .      .      ..9.3 

Green  manure 8.0 

Beijerinck's  mannite  solution 5.6 

After  humification,  these  substances  were  even  more  readily 
assimilated  and  the  nitrogen-fixation  was  greater  than  when  the 
unhumified  substance  was  used. 

The  same  year  Hanzawa  published  results  which  show  that  stable 
manure  even  up  to  3  per  cent,  greatly  stimulated  bacterial  activities. 
Green  manure  humus  was  found  by  him  to  be  injurious.  From  this 
it  is  certain  that  humus  can  act  as  a  source  of  energy  and  usually 
stimulates  bacteria,  but  the  extent  is  governed  largely  by  its  com- 
position and  by  the  quantity  of  available  combined  nitrogen  which 
is  being  supplied  with  it  to  the  organism.  In  addition  to  this,  corn 
roots,  cornstalks,  oak  leaves,  lupine,  alfalfa,  maple  leaves,  and  pine 
needles  may  all  serve  as  a  useful  source  of  energy  to  the  nitrogen- 
fixing  organisms.  Apparently,  the  tissues  from  the  non-legume 
give  a  greater  gain  than  do  those  from  the  legumes.  Fulmer  has 
recently  confirmed  these  results. 

The  influence  of  stable  manure  upon  the  nitrogen-fixing  powers 
of  the  soil  under  field  conditions  is  seen  from  the  following  table  in 
which  the  quantity  of  nitrogen  fixed  in  the  unmanured  soil  has  been 
taken  as  100  per  cent. 


METABOLISM  OF  AZOTOBACTER  267 

Treatment.  Nitrogen  fixed. 

per  cent. 

No  manure 100 

5  tons  of  manure  per  acre 103 

10  tons  of  manure  per  acre    .......  .  110 

1 5  tons  of  manure  per  acre .  105 

20  tons  of  manure  per  acre ....  103 

25  tons  of  manure  per  acre 101 

These  results  indicate  clearly  that  stable  manure  has  a  beneficial 
effect  upon  the  nitrogen-fixing  powers  of  the  soil,  but  if  used  in 
large  quantities  the  benefit  is  not  so  pronounced  as  if  used  in  smaller 
quantities. 

This  decrease  in  nitrogen-fixation  with  increased  additions  of 
manure  must  be  considered  as  due  to  its  physical  effect  upon  the 
soil,  for  Richards  found  that  Azotobacter  grow  and  fix  nitrogen  in 
horse  manure  when  it  is  well  aerated  and  contains  sufficient  moisture 
and  calcium  carbonate.  There  is,  too,  a  close  connection  between 
the  diet  and  the  effect.  Horses  fed  on  oats  gave  f eces  which  induced 
the  greatest  fixation;  horses  on  grass  next;  cattle  receiving  oatmeal 
cake  third;  but  the  feces  from  cattle  fed  on  grass  proved  unsuitable. 

Manures  often  contain  nitrogen-fixing  organisms  of  considerable 
activity.  Their  activity  appears  to  be  greatest  in  fermenting  ma- 
nures mixed  with  straw  which  serves  as  a  source  of  energy. 

Although  Fulmer  and  Fred  were  unable  to  find  Azotobacter  in  any 
of  the  samples  of  manure  examined,  they  did  obtain  many  nitrogen- 
fixing  bacteria  from  it.  One  of  these  organisms,  for  which  they 
suggested  the  name  of  B.  azophile,  is  as  efficient  in  fixing  nitrogen 
as  is  Azotobacter.  This  would  make  it  appear  that  manure  may  often 
carry  to  the  soil  nitrogen-fixing  organisms. 

Metabolism  of  Azotobacter.— Much  time  has  been  given  to  a  study 
of  the  metabolism  of  Azotobacter,  yet  our  knowledge  of  this  subject 
is  far  from  satisfactory.  It  is  well  known  that  the  organisms  oxidize 
the  various  carbohydrates  and  with  the  energy  thus  obtained  build 
up  complex  nitrogen  compounds.  Berthelot  early  recognized  that 
the  nitrogen  so  fixed  is  insoluble  in  water,  thus  indicating  its  protein 
nature.  Lipman  found  that  there  was  a  small  but  appreciable 
quantity  of  nitrogen  in  both  young  and  old  cultures  of  A.  mnelandii 
not  precipitated  by  lead  acetate  and  a  large  proportion  not  precipi- 
tated by  phosphotungstic  or  by  tannic  acid.  Further  work  indicated 
that  the  substances  were  either  amino-acids  or  comparatively  simple 
peptids.  He  considered  that  one  of  the  early  substances  synthesized 
by  these  organisms  is  alaniri.  An  analysis  of  the  Azotobacter  mem- 
brane gave  the  following  results: 

Nitrogen  as  Non-Basic  Nitrogen  in  MgO       Total  per  cent. 

Ammonia  Basic  Nitrogen  Nitrogen  Precipitate  Nitrogen 

per  cent.  per  cent.  per  cent.  per  cent.  per  cent. 

0.98  2.76  6.39  0.42  10.45 


268  AZOFICATION 

This  he  finds  corresponds  remarkably  closely  with  that  of  legumin. 
Experiments  with  plants  indicate  that  the  nitrogen  of  the  Azoto- 
bacter  cells  is  not  readily  assimilated. 

Stoklasa  found  the  Azotobacter  cells  to  contain  10.2  per  cent,  ot 
total  nitrogen  and  8.6  per  cent,  of  ash.  The  ash  contained  from  58 
to  62.35  per  cent,  of  phosphoric  acid.  Nitrogen  and  phosphorus 
were  mainly  in  the  form  of  nucleo-proteins  and  lecithin.  The  per- 
centage of  both  nitrogen  and  phosphorus  in  the  cell  increase  with 
age. 

The  most  complete  analyses  of  the  Azotobacter  cells,  so  far  reported, 
show  them  to  contain  when  grown  on  dextrin  agar  and  rapidly 
dried  at  30°  C.,  6.63  per  cent,  of  water,  4.12  per  cent,  of  ash,  and 
12.92  per  cent,  of  protein.  The  protein  is  similar  to  other  plant 
proteins.  It  contains  10  per  cent,  of  ammonia  nitrogen,  26.5  per 
cent  of  diamino-nitrogen,  and  60  per  cent,  of  monoamino-nitrogen. 
The  quantity  of  lysin  present  is  very  high,  but  the  histidin  is  present 
only  in  traces. 

Krzemieniewski  states  that  Azotobacter  produces  no  hydrogen  or 
other  combustible  gases  in  its  metabolism,  but  according  to  Stoklasa 
it  does,  and  in  the  presence  of  nitrates  it  produces  ammonia  and 
nitrites.  Moler  claims  that  during  its  life,  A.  chroococcum  separates 
no  soluble  compounds,  and  it  is  only  after  death  that  it  furnishes 
nitrogen  to  higher  plants.  Nor  are  their  bodies  readily  broken 
down  by  proteolytic  enzymes.  Both  A.  agilis  and  A.  vinelandii 
separate  a  soluble  compound.  The  protein  compounds  so  formed 
in  soil  are  quickly  broken  down  by  other  bacteria.  Remy  considers 
the  nitrogen  fixed  by  Azotobacter  in  a  readily  available  form  for 
plant  assimilation.  Beijerinck  found  that  50  per  cent,  of  the  total 
nitrogen  in  Azotobacter  cells  when  supplied  to  the  soil  is  nitrified  in 
about  seven  weeks.  None-eLthe  Azotobacter  so  far  studied  produce 
nitrates  in  the  media. 

^  Turning  now  to  the  breaking  down  of  the  carbohydrates,  we  find 
that  the  organisms  produce  ethyl  alcohol,  glycocoll,  acetic  acid, 
butyric  acid,  lactic  acid,  carbon  dioxid  and  hydrogen.  The  quantity 
and  quality  of  the  different  products  vary  with  the  species  and  with 
the  carbohydrate  used. ) 

It  is  likely  that  many  of  the  end-products  have  not  been  deter- 
mined, for  Stoklasa  starting  with  15.9  gin.  of  dextrose  recovered 
7.9  as  carbon  dioxid,  0.3  as  ethyl  alcohol,  0.2  as  formic  acid,  0.7  as 
acetic  acid,  0.2  as  lactic  acid,  but  could  not  trace  the  remaining  6.6 
gm.  The  organisms  are  extremely  active  when  growing  under 
appropriate  conditions,  for  1  gm.  weight  of  Azotobacter  has  evolved 
no  less  than  1.3  gm.  of  carbon  dioxid  in  twenty-four  hours.  A  great 
distinction  between  the  Azotobacter  and  the  other  species  is  that  the 
former  decompose  their  sugar  with  carbon  dioxid  as  the  chief 


METABOLISM  OF  AZOTOBACTER  269 

product,  whereas  the  other  species  produce  large  quantities  of  butyric 
acid.    Some  of  these  products  may  be  accounted  for  as  follows : 

CHO  CHO  CHO  CHO  -»HCOOH  ->  CO2 

I  I  I  I    

CHOH          COH  CO          — >  CO          -+  CHO         ->  CH2OH 

I  I!  I  I  I  -I 

CHOH   ->  CH          ->  CH2  CH3  CH3  CH3 

I  I  I  

CHOH    CHOH    CHOH  ->  CHO     CHO      CHO 

I      I      I      I      I      ! 

CHOH    CHOH    CHOH    CHOH  ->  C— OH  -*  CO 

I  I  I  I  II  ! 

CH2OH        CH2OH         CH2OH         CH2OH         CH2  CH3 


It  is  known  that  when  sugars,  such  as  glucose,  levulose  and  man- 
nose  are  acted  upon  by  alkalies,  there  are  produced  a  great  many 
products,  some  of  which  are  formic,  carbonic,  oxalic,  lactic,  pyruvic 
tartronic,  malic,  malonic,  tartaric,  ribonic,  saccharic,  and  gluconic 
acids  in  addition  to  many  other  either  more  or  less  complex  com- 
pounds. We  can  readily  conceive  that  the  Azotobacter  brings  about 
a  somewhat  similar  reaction,  the  stages,  however,  being  more  nicely 
governed,  because  of  enzymes.  Many  of  the  products  would  be 
oxidized  to  carbon  dioxid  and  water  with  the  liberation  of  energy 
necessary  for  the  endothermic  nitrogen  reaction;  others  readily 
react  with  the  resulting  nitrogen  compounds.  We  are  completely  in 
the  dark  as  to  what  this  first  nitrogen  compound  is,  but  we  know  that 
the  Azotobacter  possess  the  power  of  changing  nitrates  or  nitrites 
under  appropriate  conditions  into  ammonia.  Up  to  date  it  has  been 
impossible  to  detect  nitrate  formation;  it  is  not  impossible  that 
nitrates  are  formed  and  utilized  by  intracellular  enzymes.  By  using 
nitrates,  nitrites  or  ammonia,  we  can  offer  a  rough  explanation  of 
protein  anabolism. 

The   endothermic   reaction, 

2N+2H2O  =  NH4NO2, 

may  take  place  and  the  ammonia  thus  formed  may  react  with  the 
decomposition  products  of  the  sugars,  pyruvic  acid  for  instance, 
with  the  formation  of  alanin  which  Lipman  considered  as  one  of 
the  first  products: 

CH3— CO— COOH     +     NH3     =     CH3— CHN— COOH     +     H2O 
CH3— CHN— COOH     +     H2     =     CH8— CHNH2— COOH 

or  with  glyoxylic  acid  forming  glycocoll : 

HCO— COOH     +     NH3     =     HCNH— COOH     +     H2O 
CHNH— COOH     +        H2     =     CH2NH2— COOH 

By  similar  reactions  other  amino-acids  may  be  formed.  More- 
over, Windas  and  Knoop  have  shown  that  methvlimadazol  may  be 


270  AZOFICATION 

produced  from  glucose  and   ammonia,   presumably   through   the 
formation  of  pyruvic  aldehyd  and  f  ormaldehyd : 

CH3  HN— C— CH3 

I  I    1 

CO      +     2NH3     +     HCHO      =     HC  +     3H2O 

I  I       I 

CHO  N— C— H 

which  is  nearly  related  to  the  amino-acid,[Jiistidin: 

H— N— C— CHs— CHNH2COOH 

!      II 

+     CH2NH2COOH  -»H—  C      |  |  +     H2 

II       II 
N— C— H 

The  various  amino-acids  may,  through  the  intervention  of  pro- 
teinases,  condense  with  the  formation  of  dipeptids,  thus: 

CH3— CHNH2COOH     +     CH3CHNH2COOH      = 

CH3CHNH2CONHCHCH3COOH     +     H2O 

By  the  continuation  of  this  process  and  by  condensing  with  phos- 
phorus and  sulphur-bearing  compounds,  probably  through  the 
intervention  of  other  enzymes,  there  may  result  the  complex  protein 
of  the  Azotobacter  cell. 

s  Pigment  Production  by  Azotobacter.— Most  species  of  Azotobacter 
produce  pigments.  These  vary  in  color  from  brown  to  black  of  the 
A.  chrcococcum  to  a  yellow  or  orange  of  the  A.  mnelandii.  The 
pigmented  film  usually  develops  on  the  culture  media  in  from  three 
to  seven  days.  It  is  formed  by  A.  chroococcum  earlier  and  in  more 
abundance  where  old  brownish  cultures  are  used  as  the  inoculating 
material.  The  pigment  is  produced  and  retained  within «the  bacterial 
cell;  it  occurs  in  neither  the  capsule  nor  the  medium.  The  pigment 
produced  by  A.  chroococcum  is  most  pronounced  when  a  dextrin 
agar  medium  to  which  calcium  carbonate  is  added  is  kept  at  a  tem- 
perature of  30°  C.  under  well  aerated  conditions.  According  to 
Jones,  it  is  produced  only  when  there  is  a  lack  of  suitable  available 
nutrient  material  and  when  organisms  in  the  pigmented  area  have 
ceased  to  multiply.  The  color  of  the  pigment  is  intensified  if 
nitrates  are  added  to  the  medium  in  which  the  organism  is  growing. 
The  non-pigmented  strains  apparently  fix  nitrogen  just  as  readily 
as  do  those  which  have  not  lost  the  power  of  forming  pigments. 

The  pigment  from  Azotobacter  chrcococcum  is  insoluble  in  water, 
alcohol,  ether,  chloroform,  benzol,  and  carbon  bisulphid.  It  dis- 
solves in  alkalies,  undergoing  decomposition  with  the  formation  of 
a  dark  brown  solution.  Sackett  maintains  that  the  peculiar  brown- 
ish color  which  is  characteristic  of  certain  u  nitre  spots"  of  some  soils 
is  due  to  the  pigment  produced  by  Azotobacter.  Such  soils  are  high 


MORPHOLOGY  OF  THE  NITROGEN-FIXING  ORGANISMS    271 

in  nitrates  and  alkalies  which  would  dissolve  the  pigments  from  the 
body  of  the  organism.  But  Omelianski  and  Sswewrowa  are  of  the 
opinion  that  althought  in  some  cases  the  dark  color  of  vegetable 
soil  may  be  due  in  a  measure  to  the  action  of  these  microorganisms, 
it  would  be  a  mistake  to  attribute  it  to  this  factor  alone.  Further- 
more, it  has  recently  been  proved  that  the  brown  color  of  the 
"nitre  spots"  is  due  to  solvent  and  decomposing  action  of  the 
nitrates  on  the  colored  organic  compounds  of  the  soil,  for  they  may 
be  produced  at  will  in  a  rich  greenhouse  soil  with  an  excess  of  sodium 
nitrate,  and  this  too  in  soils  which  have  been  rendered  sterile  with  a 
saturated  solution  of  mercuric  chlorid. 

Morphology  of  the  Nitrogen-fixing  Organisms.— Of  the  many 
different  bacteria  which  have  been  isolated  and  proved  to  have  the 
ability  to  assimilate  free  nitrogen,  Clostridium  pasteurianum  may  be 
taken  as  a  type  of  the  anaerobic  and  Azotobacter  chroococcum  as  a 
type  of  the  aerobic. 

Clostridium  pasteurianum  is  a  short  thick  rod  from  1.2  to  1.3  /-i 
in  diameter  and  1.5  to  2/z  long  in  the  young  cells;  the  older  spore- 
bearing  cells  take  on  a  spindle  shape.  The  bacteria  stain  a  violet 
brown  with  iodin.  The  spores  when  ripe  are  1.6/z  long  and  1.3^ 
broad  and  often  lie  in  a  roughly  triangular  covering.  The  ripe 
spore  escapes  through  the  wall  of  the  mother  in  a  longitudinal 
direction.  Their  germination  is  polar. 

;  Azotobacter  chroococcum  occurs  ordinarily  as  diplococci  or  short 
rounded  rods  1  to  2/i  thick  and  1.5  to  3/j  long,  and  according  to 
Prazmowski  the  microorganism  first  presents  itself  in  its  vegetative 
stage  as  a  bacterium,  in  the  fruiting  stage  as  a  micrococcus,  and 
possesses  a  nucleus  which  functions  in  the  same  way  as  that  of  higher 
animals.  /  In  the  resting  stage  the  nucleus  assumes  a  globular  form, 
having  a  strongly  refractive  nucleolus  with  clearly  differentiated 
boundary  layers.  The  individuality  of  the  nucleus  appears  to  be 
practically  lost  at  times,  because  of  its  relation  to  the  cytoplasm. 
The  division  of  the  nucleus  marks  the  first  stage  of  cell  division. 
According  to  Bonazzi  the  organism  shows  peculiar  granulations 
apparently  not  related  to  reproduction.  These  take  the  basic  dyes 
and  are  neither  fats,  glycogen,  starch  nor  chromatin,  but  appear  to 
be  of  metachromatic  nature  and  seem  to  have  their  genesis  in  the 
nucleus.  Their  disposition  in  the  cells  is  not  constant  but  changes 
in  different  individuals.  Involution  forms  occur  and  cell  division 
is  preceded  by  a  simple  form  of  mitosis.  Some,  but  not  all,  varieties 
have  been  observed  to  form  spores.  The  volutin  bodies  within  the 
organism  increase  in  number  and  size  when  the  organisms  are  grown 
on  media  rich  in  nitrates.  Hills  suggests  that  they  may  have  some 
relation  to  nitrogen-fixation,  but  his  results  appear  to  oppose  this 
view;  whereas  the  addition  of  nitrates  to  a  medium  greatly  increased 
the  reproduction,_it  very  materially  decreased  the  physiological 


272  AZOFICATION 

efficiency  of  the  organism.  It  seems,  therefore,  more  likely  that 
they  are  reserve  protein  material. 

Lohnis  and  Smith  have  recently  observed  that  Azotobacter,  in 
common  with  many  other  bacteria,  pass  through  a  life  cycle  which 
is  not  less  complicated  than  those  of  other  microorganisms.  Under 
certain  conditions  they  pass  over  into  an  amorphous  or  "  symplastic" 
stage,  appearing  under  the  microscope  either  as  an  unstainable  or  a 
readily  stainable  mass  without  any  easily  distinguishable  organiza- 
tion, which,  if  not  discarded  as  dead,  later  gives  rise  to  new  regenera- 
tive forms.  They  multiplsAnot  only  by  fission,  but  by  the  formation 
of  gonidia. 

Methods.— Clostridium  pasteurianum  grows  readily  in  a  vacuum  on 
carrots.  The  organism  also  grows  on  sliced  potatoes,  but  ordinarily 
is  grown  in  an  aqueous  solution  containing  1  gm.  K3PO4,  0.5  gm. 
MgSO4,  0.1  to  0.02  gm.  NaCl,  FeSO4,  and  MnSO4,  and  1.0  gm. 
CaCO3,  and  10  to  15  gms.  of  a  suitable  carbohydrate  in  1  liter  of 
water.  One  method  used  by  Winogradsky  in  isolating  B.  Clostridium 
pasteurianum  was  to  add  garden  soil  to  a  non-nitrogenous  solution 
and  to  allow  a  stream  of  nitrogen  gas  to  pass  through  the  solutions, 
after  which  several  successive  transfers  were  made  into  similar 
media.  The  final  culture,  after  B.  Clostridium  pasteurianum  had 
formed  spores,  was  heated  to  80°  C. 

The  organism  ferments  certain  carbohydrates  with  the  formation 
of  butyric  acid,  acetic  acid,  carbon  dioxid,  and  water.  When  grown 
in  nutritive  solution  devoid  of  combined  nitrogen,  it  assimilates 
atmospheric  nitrogen.  Although  in  pure  cultures  it  is  an  anaerobe, 
in  impure  cultures  it  may  fix  nitrogen  under  aerobic  conditions.  In 
nature  it  occurs  in  connection  with  two  other  bacteria  which  do  not 
possess  the  power  of  fixing  nitrogen,  and  their  nitrogen  requirements 
are  small.  When  in  conjunction  with  these  organisms,  Clostridium 
pasteurianum  has  the  ability  of  growing  in  the  upper  layers  or  soil 
and  of  assimilating  free  nitrogen. 

Azotobacter  chroococcum  grows  readily  on  solid  or  liquid  media, 
one  of  the  best  being: 

Per  Cent. 
Monopotassium    phosphate    (neutralized    to    phenolphthalein    by 

Sodium  hydroxid 0.02 

Magnesium  sulphate 0.02 

Sodium  chlorid 0 . 02 

Calcium  sulphate 0.01 

Ferric  chlorid  (1  per  cent,  solution),  2  drops  per  100  c.c.  mannite  1.00 

The  organism  is  readily  isolated  by  seeding  this  medium  with 
soil.  After  the  characteristic  membrane  forms,  it  is  transferred  by 
dilution  to  a  similar  medium  containing  agar  in  which  the  charac- 
teristic brownish  black  colonies  form  readily. 

On  mannite  agar  the  colonies  first  appear  as  milk-white  glistening 
drops,  round  and  convex,  which  under  a  low  magnification  show 


METHODS  273 

a  coarsely  granular  structure  extending  to  the  margin.  The  colonies 
rapidly  increase  in  size,  and  after  a  week  or  more  become  brown  at 
the  center  with  concentric  rings  alternating  dark  and  white  to  the 
circumference  and  darker  streaks  radiating  from  the  center  outward. 
In  old  cultures,  where  the  agar  has  partly  dried  up,  the  cells  are  often 
united  in  sarcina-like  packets;  the  cell  walls  are  much  swollen  and 
the  contents  are  aggregated  to  a  small  ball  at  the  center.  At  the 
same  time  giant  cells,  both  round  and  elongated  and  filled  with  oil 
drops,  can  be  seen.  Often  a  number  of  involution  forms  are  seen, 
drawn  out  with  long  threads  and  false  septa.  By  successive 
dilutions  and  transfers,  it  may  be  obtained  in  pure  culture,  although 
at  times  considerable  difficulty  is  experienced  in  freeing  it  from  a 
small  organism—  B.  radiobacter. 

Several  different  methods  have  been  used  for  studying  its  nitro- 
gen-fixing powers : 

(a)  Seeding  into  100  c.c.  of  the  medium  given  above  and  after  a 
certain  time  determining  the  nitrogen. 

(b)  The  use  of  the  same  medium,  but  the  addition  of  sufficient 
sand  for  the  formation  of  sand  slopes  on  which  the  organism  can 
grow. 

(c)  The  addition  of  a  definite  quantity  of  a  carbohydrate  to  a 
soil  and  the  incubation  of  this. 

Each  of  these  methods  has  its  value.  The  first  is  much  more 
readily  handled  in  the  final  Kjeldahl  determination,  but  the  others 
give  much  higher  results. 

Freudenreich  found  that  when  Azotobacter  are  grown  upon  gyp- 
sum, the  gain  in  nitrogen  is  considerably  in  excess  of  that  assimilated 
in  the  liquid  media.  Krainsky  found  Azotobacter  to  utilize  from  100 
to  200  gin.  of  sugar  in  the  assimilation  of  1  gm.  of  nitrogen  when 
grown  in  solution,  but  when  grown  on  sand  it  required  only  11  to 
30  gm.  for  the  same  fixation.  Many  other  workers  have  noted 
similar  variation  when  grown  in  the  soil.  Where  the  organisms 
have  been  grown  on  gypsum  or  soil,  we  may  attribute  the  stimula- 
tion to  certain  soluble  constituents,  yet  this  explanation  scarcely 
seems  plausible  when  considered  in  relation  to  sand  cultures.  Three 
strains  of  Azotobacter  were  grown  in  Ashby's  mannite  solution 
and  sand  (nearly  pure  silicon  dioxid)  to  which  Ashby's  solution  was 
added,  with  the  following  results: 

Nitrogen  fixed  in       Nitrogen  fixed 
Ashby's  solution,  in  sand, 

mgm.  mgm. 

Azotobacter  A 6.86  22.61 

Azotobacter  B 5.00  12.60 

Azotobacter  C 6.44  16.80 

Moreover,  arsenic  is  very  toxic  in  the  solution,  whereas  when 
added  to  the  soil  or  to  pure  quartz,  in- small  quantities,  it  stimulates. 
Although  the  total  quantities  of  nitrogen  fixed  under  the  two 
18 


274  AZOFICATION 

methods  differ  greatly,  the  relative  efficiency  of  the  organisms  is 
about  the  same  in  both  cases.  In  testing  soils  similar  results  are 
obtained,  as  may  be  seen  from  the  following  results,  which  are  the 
average  for  several  hundred  determinations  made  on  different  soils 
by  the  two  methods. 

Nitrogen  fixed  in: 

f()0  c.c.  of  Ashby's 

100  gm.  of  soil  containing  1.5  gm. 

Depth  of  Sample.  1.5  gm.  of  mannite,  of  mannite 

mgm.  mgm. 

Firstfoot 5.28  2.11 

Second  foot 2.42  0.77 

Third  foot 1.55  0.58 

Although  the  greater  aeration  in  the  sand  and  soil  culture  probably 
play  a  great  part,  there  is  little  doubt  that  the  colloids  also  assist. 

Relation  of  Azotobacter  to  other  Organisms.— In  the  early  study  of 
nitrogen-fixation,  the  view  was  held  that  algae  growing  on  or  near 
the  surface  of  soil  are  able  to  fix  nitrogen.  Frank  in  1888  had 
observed  such  a  growth  on  sand  exposed  to  light  and  found  that  the 
soil  showed  a  considerable  increase  in  nitrogen.  In  1892  Schlosing 
and  Laurent  proved,  both  by  determining  the  nitrogen  fixed  by  a 
soil  in  a  closed  vessel  and  by  observing  the  diminution  of  the  nitrogen 
gas  in  the  enclosed  air,  that  a  soil  exposed  to  light  gains  in  nitrogen 
if  algae  are  allowed  to  grow  on  the  surface,  and  that  the  gain  is 
confined  to  the  upper  few  millimeters.  They  did  not,  however, 
employ  a  pure  soil  or  pure  cultures  of  algae.  Kossowitsch,  working 
with  pure  cultures  of  two  green  algae,  found  no  fixation,  but  observed 
a  considerable  increase  of  soil  nitrogen  when  they  were  grown  with 
soil  bacteria.  Later,  Kruger  and  Schneidewind,  employing  pure 
cultures  of  many  other  chlorophyceae,  obtained  no  nitrogen-fixation, 
Hellriegel  and  Deherain  had  found  a  large  increase  in  the  nitrogen 
content  of  sand  in  pots  when  exposed  to  the  light,  which  was  always 
accompanied  by  a  development  of  algae.  In  the  light  of  such  results, 
the  conclusion  has  been  reached  that  algae  alone  cannot  assimilate 
free  nitrogen,  but  only  in  concurrence  with  soil  bacteria,  the  former 
producing  carbohydrates  which  are  used  by  the  latter  as  a  source 
of  energy  for  the  nitrogen-fixation.  Heinze  actually  observed  rapid 
fixation  of  nitrogen  when  cultures  of  algae  were  inoculated  with  Azoto- 
bacter or  other  nitrogen-fixing  organisms.  Stoklasa  found  that  Azoto- 
bacter are  especially  abundant  in  soils  having  a  vigorous  growth  of 
blue-green  algae.  Azotobacter  are  often  absent  from  virgin  soils, 
but  are  always  found  in  such  soils  when  there  is  a  vigorous  growth 
of  algae.  Bottomley  claims  that  both  Azotobacter  and  Pseudomonas 
live  in  true  symbiosis  with  cycas.  It,  therefore,  appears  certain 
that  the  nitrogen-fixing  powers  of  Azotobacter  are  greatly  enhanced 
when  growing  with  algae,  but  the  exact  role  played  by  each  is  yet  to 
be  explained.  This  offers  a  rich  and  inviting  field  for  research. 

Nor  is  it  alone  in  combination  with  algae  that  these  organisms 


AZOTOBACTER  RELATION  TO  OTHER  ORGANISMS        275 

may  grow  and  thus  be  benefited.  Beijerinck  and  van  Delden  early 
recognized  that  an  apparent  symbiosis  exists  between  Azotobacter 
and  other  bacteria,  and  that  the  nitrogen  fixed  is  considerably 
greater  in  the  mixed  than  in  the  pure  cultures.  This  symbiosis, 
though  in  many  cases  beneficial  to  Azotobacter,  is  not  essential  for 
nitrogen-fixation.  Radiobacter,.  with  which  the  Azotobacter  are 
usually  associated,  have  only  slight  nitrogen-fixing  powers,  yet  they 
increase  the  nitrogen-fixing  powers  of  Azotobacter.  The  carbo- 
hydrates disappear  more  rapidly  from  mixed  than  from  pure  cultures 
and  with  a  greater  fixation  per  gram  of  carbohydrate  utilized. 
There  is  also  a  greater  fixation  when  two  strains  of  Azotobacter  are 
grown  in  conjunction  with  each  other.  This  is  especially  marked  in 
an  aqueous  solution  of  mannite.  Results  have  been  reported  where 
Azotobacter  fixed  twice  as  much  in  the  presence  of  Pseudomonas  as 
when  grown  alone. 

The  manner  in  which  this  mutual  benefit  is  exerted  is  not  clear. 
In  some  cases  it  may  be  due  to  the  associated  organism  rendering 
more  available  the  carbonaceous  material. 

Omelianski  and  Salunskov  offer  the  following  explanation  con- 
cerning the  association  of  aerobic  and  anaerobic  nitrogen-fixers: 

"The  synergetic  activity  of  nitrogen-fixing  and  accompanying 
microbes,  is  both  in  laboratory  experiments  and  under  natural 
conditions  (cultivable  stratum  of  the  soil)  of  a  different  character 
according  to  the  properties  of  the  species  taking  part  in  the  process 
and  their  environment;  in  both  cases  the  function  of  the  satellite 
organism  seems  to  consist  in  fixing  the  oxygen  of  the  air  and  creating 
the  anaerobic  environment  for  Clostridium  pasteurianum.  The 
species  added  to  the  cultures  of  nitrogen-fixing  microbes  sometimes 
supply  the  compounds  of  carbon  needed  for  the  process  of  fixing 
nitrogen  as  energetic  substance.  In  the  case  of  the  combination: 
Azotobacter  and  Clostridium  pasteurianum,  the  function  of  the  former 
is  not  confined  to  fixing  the  oxygen  of  the  air  only,  and  consequently 
to  creating  an  anaerobic  environment  for  the  Clostridium.  But  this 
combination  is  also  useful  inasmuch  as  it  destroys  the  injurious 
products  of  disassimilation  created  by  the  second  (chiefly  butyric 
acid)  and  maintains  the  action  of  the  environment.  (Azotobacter 
is  alkaligenic  and  the  Clostridium  acidogenic.) 

"The  satellite  species  may  also  unfavorably  affect  the  nitrogen- 
fixing  microbe,  either  through  products  of  assimilation  or  by  con- 
sumption of  the  carbon  compounds  needed  by  this  microbe  for 
nitrogen-fixing.  The  energetic  fixation  of  oxygen  by  the  satellite 
aerobic  species  creates  conditions  favorable  to  the  development  of 
Clostridium  pasteurianum,  but  at  the  same  time  hinders  the  growth 
of  the  Azotobacter,  which  is  necessarily  aerobic. 

"  The  form  endowed  with  the  maximum  vitality  and  at  the  same 
time  the  most  common  form  in  which  combination  of  the  nitrogen- 


276  AZOF1  CATION 

fixing  organisms  takes  place  in  the  upper  soil  strata  is  that  of  sym- 
biosis between  the  aerobic  and  anaerobic  nitrogen  fixers,  principally 
between  Azotobacter  and  Clostridium  pasteurianum.  In  spite  of  the 
opposite  properties  of  the  two  species,  their  synergetic  activity  in 
the  upper  strata  of  the  soil  results  in  a  harmonious  mutual  develop- 
ment producing  the  maximum  economy  in  consumption  of  energetic 
substances." 

So  far,  little  has  been  done  to  determine  the  relationship  of 
Azotobacter  to  the  higher  plants,  but  it  is  interesting  to  note  that 
Beijerinck  has  observed  a  distinct  relationship  between  the  distribu- 
tion of  the  organism  and  leguminous  plants.  Fischer  suggests  that 
some  nitrogen-fixing  bacteria  presumably  exist  first  as  saprophytes, 
then  as  exoparasites  in  loose  combination  with  green  plants,  then  as 
endoparasites.  Finally  they  develop  the  true  symbiosis  or  root 
nodule  bacilli.  Hopkins  has  questioned  whether  there  may  not  be  a 
relationship  between  the  legume  bacteria  and  Azotobacter. 

The  Influence  of  Water.— ^Azotobacter  are  very  resistant  to  drying; 
they  may  be  dried  for  a  considerable  time  in  a  desiccator  over  sul- 
phuric acid.  Pure  cultures  are  just  as  resistant  to  drying  as  are  mixed 
cultures. )  This  would  vary  some  with  the  media  in  which  the 
bacteria  are  dried,  for  the  survival  of  non-sporebearing  bacteria  in 
air-dry  soil  is  due,  in  part,  to  the  retention  by  the  soil  of  moisture 
in  the  hygroscopic  form.  This,  however,  is  not  the  only  factor,  for 
the  longevity  of  bacteria  in  a  solid  is  not  directly  proportional  to  its 
grain  size  and  hygroscopic  moisture.  <  Giltner  and  Langworth  found 
that  bacteria  resisted  desiccation  longer  in  a  rich  clay  loam  than  in 
sand.  Furthermore,  if  bacteria  are  suspended  in  the  extract  from 
a  rich  clay  loam  before  being  subjected  to  desiccation  in  sand,  they 
live  longer  than  if  subjected  to  dessiccation  after  suspension  in  a 
physiological  salt  solution.  Because  of  this,  they  consider  that  soils 
contain  substances  which  have  a  protective  influence  upon  bacteria, 
subject  to  desiccation.  t 

Lipman  and  Burgess  have  found  that  many  soils  manifest  a  vigor- 
ous nitrogen-fixing  power  even  after  being  air-dried  and  kept  in 
stoppered  museum  bottles  for  periods  varying  from  five  to  twenty 
years.  In  some  cases  the  fixation  was  equally  as  high  as  in  freshly- 
collected  samples.  The  organisms  from  such  soils  are  more  easily 
attenuated  than  are  other  organisms  which  have  not  been  so  dried. 
The  tendency  is  for  soils  gradually  to  decline  in  nitrogen-fixing 
power  or  drying.  This  may  manifest  itself  as  early  as  the  second 
week. 

x  During  the  periods  of  drying,  the  organisms  are  inactive,  as  they 
require  moisture  for  growth  and  reproduction.  For  maximum 
nitrogen-fixation  a  definite  moisture  content  is  needed.  Warmbold 
found  the  optimum  moisture  content  to  be  20  per  cent.  ,  When  it  was 
below  10  per  cent,  there  was  no  nitrogen  fixed,  and  in  some  cases 
there  was  a  decided  loss  of  nitrogen,  Krainsky  allowed  soil  with 


Tttti  INFLUENCE  of  WATM  27? 

varying  moisture  content  to  stand  for  some  time  and  then  inoculated 
it  into  mannite  solutions  and  obtained  maximum  fixation  in  the  soils 
containing  fairly  small  quantities  of  water.  Later,  however,  he 
decided  that  soil  should  be  damp— but  not  wet— and  well  aerated 
for  maximum  nitrogen-fixation.  <  The  water  requirements  vary  with 
different  soils.  As  a  general  rule,  the  higher  the  humus  content  of 
the  soil,  the  more  water  will  be  required  for  optimum  nitrogen- 
fixation.  The  quantity  of  water  present  may,  however,  become  so 
great  that  it  may  kill  all  Azotobacter  in  addition  to  stopping  nitrogen- 
fixation.  * 

<  An  insufficient  supply  of  moisture  checks  both  nitrification  and 
nitrogen-fixation.  This  occurs  in  some  soils  when  the  water  content 
has  been  reduced  to  16.5  per  cent.  This  again  varies  with  the  soil, 
for  Schlosing  found  bacterial  activity  less  in  fine-grained  soils  than 
in  lighter,  coarse-grained  soils.>  A  difference  in  moisture  content 
of  1  per  cent,  according  to  Dafert  and  Bellinger,  is  sufficient  to  pro- 
duce a  marked  change  in  the  oxidation  going  on  in  the  soil. 

The  moisture  requirement  of  the  nitrogen-fixing  bacteria,  accord- 
ing to  Lipman  and  Sharp,  is  more  nearly  that  of  the  ammonifying 
than  of  nitrifying  organisms.  In  a  sandy  loam  it  was  found  to  vary 
between  20  and  24  per  cent,  the  anaerobic  nitrogen-fixers  are  most 
active,  but  the  action  of  the  aerobes  is  slightly  depressed.  Thus, 
in  many  soils  two  maxima  of  nitrogen-fixation  occur,  depending  upon 
whether  the  conditions  are  favorable  for  the  anaerobic  or  aerobic 
organisms. 

Traaen's  results  differ  from  Lipman' s  in  showing  only  the  one 
maximum,  as  is  seen  from  the  following,  which  gives  the  milligrams 
of  nitrogen  fixed  in  100  gm.  of  soil. 

5  per  cent.    10  per  cent.  17.5  per  cent.     25  per  cent.      30  per  cent. 
Temperature.  H2O.  H2O.  H2O.  H2O.  H2O. 

13°C 0.1  1.5  11.2  13.4  5.4 

25°C 1.9  1.9  13.2  16.6  15.5 

He  used  a  loam  soil  with  a  maximum  water  capacity  of  27.4  per 
cent.  It  is  quite  evident  from  his  statement  that  anaerobic  organ- 
isms played  a  prominent  part  in  the  fixation  at  the  higher  moisture 
contents. 

Since  the  carbohydrates  disappeared  much  more  rapidly  in  the 
soils  containing  the  greater  quantities  of  water,  it  is  quite  possible 
that  greater  quantities  of  nitrogen  per  gram  of  carbohydrate  con- 
sumed are  fixed  where  the  smaller  quantities  of  water  are  applied. 
This,  together  with  the  different  methods  used  by  the  several 
investigators,  would  explain  the  apparent  discrepancy  in  their 
results. 

In  a  series  of  pot  experiments  in  which  a  calcareous  loam  receiving 
various. amounts  of  water  was  used,  the  author  found  the  moisture 
content  for  maximum  nitrogen-fixation  to  lie  between  15  and  22  per 
cent.  These  results  also  bring  out  the  two  maxima  which  were  first 


278 


AZOFICATION 


noted  by  Lipman.  These  soils  were  kept  at  the  various  moisture 
contents  for  four  months.  All  were  then  incubated  at  28°  C.  for 
twenty-one  days  with  a  moisture  content  of  20  per  cent. 


Treatment. 
Per  cent. 

12.5 
15.0 
17.5 
20.5 
22.5 


Nitrogen  fixed. 
Per  cent. 

100 
108 
102 
104 
108 


In  this  soil  the  optimum  for  the  aerobes  would  appear  to  be  at 
17.5  per  cent,  and  that  for  the  anaerobes  22.5  per  cent,  or  higher. 


FIG.  34. — Average  percentages  of  ammonia 


and  nitric  nitrogen 


formed  and  nitrogen  fixed in  soil  receiving  varying  quantities  of  water.     On 

the  ordinate  is  given  the  per  cent,  increase  of  the  respective  substances  and  on  the 
abscissa  the  quantity  of  water  applied  as  per  cent,  of  water-holding  capacity. 


TEMPERATURE  279 

When  too  large  a  quantity  of  water  is  applied  there  is  a  tendency 
to  depress  the  total  nitrogen  fixed,  as  is  illustrated  by  the  following 
results  in  which  various  quantities  of  water  were  applied  to  a  soil 
throughout  the  year  under  field  conditions : 

Inches  of  Nitrogen  fixed 

water  applied  in  100  grams 

during  summer.  soil. 

mgm. 

37.5 1.4 

25.0 2.1 

15.0 8.5 

None  .  3.5 


The  maximum  for  anaerobic  conditions  does  not  appear  in  these 
results  probably  because  the  soil  did  not  become  filled  with  water 
and  because  under  field  conditions  the  water  rapidly  drains  away 
or  is  evaporated.  There  would  seem  to  be  a  correlation  between 
the  water  content  of  a  soil  as  measured  in  terms  of  its  water-holding 
capacity  irrespective  of  physical  composition  and  its  nitrogen-fixing 
powers.  This  is  brought  out  in  Fig.  34  in  which  water  requirements 
for  ammonification,  nitrification,  and  nitrogen-fixation  are  compared. 

Temperature. — Berthelot  early  recognized  that  the  biological  gain 
of  nitrogen  in  soils  is  dependent  upon  a  suitable  temperature. 
He  found  nitrogen-fixation  to  occur  best  at  summer  temperatures 
between  50°  and  104  F°.  The  process  was  immediately  stopped  on 
heating  to  230°  F.  Later  Thiele  maintained  that  although  Azoto- 
bacter  possess  the  ability  to  fix  small  quantities  of  nitrogen  under 
laboratory  conditions,  the  temperature  would  be  unfavorable  under 
field  conditions.  Heinze,  however,  found  that  although  the  nitro- 
gen-assimilating organisms  are  most  active  at  a  temperature  between 
20°  C.  and  30°  C.,  they  nevertheless  fix  appreciable  quantities  at 
temperatures  as  low  as  8  to  10°  C.  Still  more  recent  work  has  showrn 
the  optimum  temperature  to  be  28°  C.  and  the  limits  of  activity  of 
Azotobacter  chroococcum  to  lie  between  9°  C.  and  33°  C.  The  actual 
quantitative  variation  in  nitrogen  fixed  is  .seen  from  the  results 
reported  by  Lohnis.  He  inoculated  100  c.c.  of  a  1  per  cent,  mannite 
soil  extract  with  10  gm.  of  soil  and  obtained  the  following  fixation 
at  the  various  temperatures: 

Nitrogen. 
Mgm. 

10°  to  12°  C 3.15 

20°  to  22°  C 4.55 

30°  to  32°  C 4.27 

Better  fixation  at  a  lower  temperature  is  noted  wrhen  the  soil  is 
incubated  and  the  gain  in  nitrogen  determined  directly.  Koch 
obtained  fixations  of  3  mgm;,  11  mgm.,  and  15.5  mgm.  of  nitrogen 
in  100  gm.  of  soil  when  incubated  with  a  carbohydrate  at  7°  C., 


280  A20F1CAT1ON 

15°  C.,  and  24°  C.,  respectively.  Traaen,  using  a  loam  soil  with  a 
maximum  water-holding  capacity  of  27.4  per  cent.,  obtained  nearly 
as  great  a  fixation  at  13°  C.  as  at  25°  C.  when  the  optimum  moisture 
content  was  maintained.  This  is  seen  from  the  following: 

Nitrogen  fixed  in  100  gm.  of  soil. 
Temperature. 


5  per  cent. 
H,0. 

10  per  cent. 
H20. 

17.5  per  cent. 
H20. 

25  per  cent. 
H20 

30  per  cent. 
H20. 

Mgm. 

Mgm. 

Mgm. 

Mgm. 

Mgm. 

.     0.1 

1.5 

11.2 

13.4 

5.4 

.      1.9 

1.9 

13.2 

16.6 

15.5 

13°  C.    . 
25°  C.    . 

A  temperature,  favorable  even  though  not  ideal  for  nitrogen- 
fixation,  would  occur  in  soils  under  natural  conditions.  The 
temperature  of  soil  in  Utah  during  the  months  if  September  averaged 
14°  C.,  with  a  minimum  of  10°  C.  and  a  maximum  of  17°  C.  During 
June,  July  and  August  the  mean  temperatures  would  be  much 
higher. 

The  mean  daily  temperatures  of  the  soil  for  Bismarck,  North 
Dakota;  Key  West,  Florida;  and  New  Brunswick,  New  Jersey;  for 
the  months  of  June,  July,  August  and  September  were  18°  C.,  28°  C., 
and  24.5°  C.,  respectively.  From  this  it  is  evident  that  during  a 
considerable  period  of  each  year  an  arable  soil  has  a  temperature  high 
enough  for  moderately  rapid  nitrogen-fixation. 
^Although  it  is  generally  maintained  that  there  is  no  nitrogen- 
fixation  in  soils  during  the  winter  months,  cold  or  even  freezing  does 
not  injure  the  organism;  for  the  cooling  of  a  soil,  even  to  the  freezing 
point,  increases  its  nitrogen-fixing  powers.  This  is  probably  due  to 
the  suppression  of  competing  species  and  to  the  establishment  of  a 
new  flora.  >  The  same  is  true  when  the  soil  is  heated,  as  may  be  seen 
from  the  results  given  below. 

Temperature  Nitrogen  fixed, 

deg.  C.  per  cent. 

Normal  5.11 

50  9.00 

55  14.14 

60*  16.38 

65  14.42 

70  13.02 

75  11.34 

80  12.66 

85  10.36 

This  sbil  had  been  autoclaved  and  then  inoculated  with  a  soil 
extract  which  had  been  heated  to  the  temperature  indicated.  The 
stimulation  could  not,  therefore,  have  been  due  to  the  heat  rendering 
more  of  the  plant-food  in  the  soil  available.  The  results  indicate 
that  many  of  the  organisms  which  take  part  in  nitrogen-fixation 
are  highly  resistant  to  heat.  It  is  significant  that  the  greatest  stimu- 
lation is  exerted  in  a  soil  which  had  been  inoculated  with  solutions 


SEASON  28l 

heated  just  above  the  temperature  which  Cunningham  and  Lohnis 
found  to  be  the  thermal  death-point  of  soil  protozoa. 

Light  and  other  Rays.— As  a  class,  bacteria  are  sensitive  to  light, 
but  the  extent  to  which  they  can  withstand  it  varies,  among  other 
things,  with  the  conditions  of  exposure  and  the  specific  organism. 
Unfortunately,  we  have  but  fragmentary  information  concerning  the 
effect  of  light  upon  azofiers,  but  what  we  do  know  would  lead  us  to 
believe  they  are  more  resistant  than  many  microorganisms— prob- 
ably more  so  than  many  other  soil  bacteria.  Berthelot  recognized 
that  nitrogen-fixation  in  the  soil  occurred  both  in  daylight  and  in 
darkness,  though  more  freely  in  the  light.  Jones  found  many 
Azotobacter  to  be  alive  in  a  small  Petri  dish  of  dried  soil  that  had 
stood  in  the  laboratory  in  front  of  a  south  window  for  two  years. 
They  can  withstand  the  direct  action  of  the  violet  and  of  the  longer 
ultraviolet  rays  for  five  minutes,  but  are  killed  in  much  less  time  by 
the  shorter  ultraviolet  rays.  They  are  more  resistant  even  to  these 
than  are  many  other  species. 

The  fixation  of  elementary  nitrogen  by  A.  chroococcum  is  distinctly 
increased  when  the  air  is  activated  by  pitchblende.  Somewhat 
better  results  are  obtained  with  weak  than  with  stronger  radio- 
active intensity. 

Aeration.— Under  field  conditions  there  is  a  mixed  flora  consisting 
of  the  anaerobic  and  aerobic  nitrogen-fixing  microorganisms.  A 
soil  condition  which  would  be  ideal  for  one  species  might  be  unfavor- 
able for  the  other.  It  has  already  been  pointed  out  that  there  are 
two  maxima  of  nitrogen-fixation  in  soils,  depending  upon  the 
moisture  content.  This  is  illustrated  in  Figure  32. 

'Although  it  is  usually  conceded  that  nitrogen-fixation  is  most 
rapid  when  soils  are  well  aerated,  this  may  not  always  be  the  case.  > 
Concerning  this  Murray  reports  the  following  results: 

Nitrogen  fixed 


Kind  of  soil.  Aerobic  Anaerobic 

conditions.  condition, 

mgm.  mgm. 

Greenhouse  soil 0.84  8.50 

Loam  soil 3.08  5.29 

Clay  soil 0.84  4.69 

*  This  condition  must  be  attributed  to  a  great  difference  in  the 
physiological  efficiency  of  the  two  groups  found  in  these  particular 
soils  and  not  to  a  lack  of  aerobic  nitrogen-fixing  organisms,  for  more 
than  ten  times  the  number  of  organisms  developed  on  nitrogen- 
poor  media  from  these  soils  under  aerobic  as  under  anaerobic  condi- 
tions. > 

Season.— Berthelot  was  unable  to  show  any  gain  in  nitrogen  of 
his  soils  during  the  winter,  but  Koch  found  a  considerable  increase 
during  this  season  in  soils  which  were  kept  in  a  heap  and  shovelled 


282  AZOFICATION 

over  from  time  to  time.  Lohnis  observed  that  Azotobacter  mem- 
branes are  more  readily  obtained  in  winter  than  in  summer.  He 
later  found  that  the  nitrogen-fixing  power  of  soil  varies  from  month 
to  month  throughout  the  year,  there  being  two  maxima — one  in 
spring  and  another  in  autumn.  The  extent  of  the  variation  noted 
may  be  seen  from  the  following: 

1903-1904:   March 100 

«            May 121 

"             July 50 

September 100 

1907:  April 100 

"       May-June 133 

"       July-August 69 

"       October-November 122 

The  relative  numbers  are  based  on  the  spring  months  as  100. 

Green  found  nitrogen-fixation  in  1  per  cent,  mannite  solution  to  be 
low  during  August,  September  and  April.  In  other  months  he 
noted  a  fairly  constant  fixation  of  about  10  mgm.  of  nitrogen  per 
gram  of  mannite.  He  also  noted  a  marked  yearly  variation  in  the 
nitrogen  fixed  during  July  and  August. 

Walton  found  nitrogen-fixation  lowest  in  Indian  soil  between 
October  and  January  and  highest  between  June  and  September. 
This  corresponds  with  moisture  and  temperature  changes.  Peterson 
has  found  that  although  the  nitrogen-fixation  of  Utah  soils  is  highest 
from  June  to  September,  the  number  of  types  of  Azotobacter  occur- 
ring in  the  soil  was  greatest  in  May.  Moll  goes  so  far  as  to  maintain 
from  his  work  that  the  season  of  the  year  is  the  principal  factor  in 
determining  the  biochemical  transformation  in  soils.  This  would 
appear  to  be  especially  true  as  regards  nitrogen-fixation. 

Crop.— Heinze  called  attention  to  the  fact  that  the  fallowing  of  the 
soil  increased  its  nitrogen-fixing  power.  This  could  be  due  to  better 
aeration,  moisture,  temperature,  etc.,  and  not  to  any  depressing 
influence  exerted  directly  by  the  plant.  Most  experiments  which 
deal  with  plant  and  bacterial  activity  could  be  interpreted  in  this 
light.  Hiltner  maintained  that  the  free  nitrogen-fixing  bacteria 
are  stimulated  in  their  activities  by  the  growing  plant  roots.  There 
may  be  considerable  truth  in  this,  for  here  the  higher  plants  are 
rapidly  removing  from  the  solution  the  soluble  nitrogen  compounds. 
In  this  case,  the  nitrogen-fixing  organisms  would  be  forced  either  to 
compete  with  the  higher  plant  for  the  soil  nitrogen  or  else  to  make 
use  of  their  ability  to  live  upon  the  atmospheric  nitrogen.  It  is 
certain  that  different  cultural  methods  vary  sufficiently  with  crops 
to  influence  profoundly  a  soil's  nitrogen-assimilating  properties, 
for  the  Azotobacter  occur  more  widely  distributed  in  cultivated  than 
in  virgin  soil.  The  analyses  of  hundreds  of  samples  of  cultivated 
and  virgin  soils  in  Utah  have  in  nearly  every  case  shown  the  virgin 
soil  to  have  a  low  nitrogen-fixing  power  as  compared  with  the  culti- 


CLIMATE  283 

vated  soil.  This  was  the  case  even  where  the  soil  was  incubated 
without  carbohydrates  and  the  nitrogen  determined  directly.  The 
average  results  for  many  determinations  were  as  follows: 

Mgm.  of 
nitrogen  fluid. 

Virgin  soil 6.99 

Cultivated 14.28 

Wheat 11.83 

Alfalfa • 12.24 

Fallow 22.81 

The  fallow  soil  had  received  considerable  manure,  hence  these 
results  are  undoubtedly  high.  It  would,  however,  be  possible  to 
fallow  or  crop  soils  so  continuously  that  extremely  small  quantities 
of  plant  residues  would  be  returned  to  the  soil,  under  which  condi- 
tions there  might  be  a  decrease  in  nitrogen-fixation.  The  conditions 
of  moisture  and  aeration  are  much  more  nearly  ideal  in  a  fallow  soil 
than  in  a  cropped  soil.  It  is  just  possible  that  the  high  fixation 
noted  where  wheat  is  grown  continuously  may  be  due  to  the  method 
in  vogue  in  the  arid  districts  of  leaving  the  greater  part  of  the  straw 
on  the  soil.  This  would  act  as  readily  assimilable  carbonaceous 
material  for  the  Azotobacter.  Welbel  and  Winkler  have  found  that 
fallowing  not  only  increases  the  assimilable  nitrogen  but  also  the 
available  phosphorus  of  the  soil,  a  liberal  supply  of  which  causes  the 
Azotobacter  to  utilize  its  energy  more  economically.  That  the 
increased  nitrogen-fixation  noted  when  soils  are  cultivated  is  not 
confined  to  the  arid  soil,  is  seen  from  the  recent  work  of  Reed 
and  Williams.  Brown's  work  indicates  that  crop  rotation  increases 
the  nitrogen-fixing  powers  of  a  soil. 

Climate.— It  has  been  maintained  for  a  long  time  that  there  is  a 
close  correlation  between  the  chemical,  physical,  and  biological 
transformations  going  on  in  a  soil  and  the  climatic  conditions,  but 
there  was  nothing  definite  on  this  subject  until  the  highly  interesting 
work  of  Lipman  and  Waynick  appeared.  They  found  a  definite 
relationship  between  climate  and  the  nitrogen-fixing  powers  of  a  soil. 
Removal  of  California  soil  to  Kansas  increased  the  vigor  of  the 
Awtobacter  flora  and  especially  that  of  A.  chroococcum.  It  increased 
the  nitrogen-fixation  by  50  per  cent,  over  that  attained  by  the  same 
soil  in  California.  Similar  results  were  obtained  in  California  soils 
removed  from  Maryland.  Kansas  soil  taken  to  California  lost  its 
power  to  produce  a  membrane  in  mannite  solution,  the  Azotobacter 
flora  became  rather  feeble,  and  the  nitrogen-fixing  powers  of  the  soil 
were  greatly  reduced.  The  removal  of  the  Kansas  soil  to  Maryland 
increased  the  vigor  of  the  Azotobacter  and  induced  a  higher  fixation 
of  nitrogen.  The  Maryland  soil  in  California  diminishes  in  nitrogen- 
fixing  powers,  but  not  in  so  great  a  degree  as  does  the  Kansas  soil. 
This  also  happened  when  the  Maryland  soil  was  taken  to  Kansas. 


284 

The  bacterial  flora  of  a  soil,  therefore,  is  dependent  upon  climatic 
conditions  which  affect  many  of  the  other  properties  of  a  soil. 

Relationship  of  Azotobacter  to  Nitrate  Accumulations.— The  fact 
that  certain  spots  in  western  cultivated  soils  were  very 
nitrates  was  first  observed  by  Hilgard  This  he  attributed 
rapid  nitrification  of  the  organic  matter  of  the  soil  in  the  warm  arid 
climate  of  the  West  when  the  moisture  limit  was  removed  by 
irrigation. 

A  number  of  years  later  Headden  noted  these  "nitre  spots"  in  a 
number  of  Colorado  soils,  but  he  attributed  it  to  the  fixation  of 
atmospheric  nitrogen  by  the  non-symbiotic  bacteria  which  find  in 
the  western  soils  ideal  conditions  for  growth  and  rapid  nitrogen 
fixation.  This  conception  has  been  further  amplified  by  Headden 
and  also  Sackett.  In  the  early  work  by  Headden  it  is  assumed 
that  the  Azotobacter  not  only  fix  the  nitrogen  but  also  produce  the 
nitrates.  It  is  known,  however,  that  these  organisms  do  not  produce 
nitrates. 

Moreover,  there  are  a  number  of  other  vital  objections  to  this 
theory.  (1)  Lipman  has  shown  that  for  the  fixation  of  the  quantity 
of  nitrogen  which  Headden  maintains  to  have  occurred,  it  would 
require  from  1000  to  2000  tons  of  carbohydrates.  There  is  no  such 
visible  supply  of  energy  in  these  soils.  True,  many  of  these  soils 
have  a  rich  algse  flora,  but  it  has  not  been  proved  that  this  will 
furnish  a  sufficient  supply  of  available  energy.  (2)  The  average 
amount  of  nitrogen  fixed  in  thirty-two  samples  collected  in  the 
nitrate  region  was  7.4  mgms.  and  the  average  nitrogen  fixed  in 
thirty-one  samples  of  (iry-farm  alkali-free  soil  in  Utah  was  12.2 
mgms.  Yet  there  is  no  accumulation  of  nitrates  in  these  latter 
soils.  (3)  The  quantity  of  soluble  salts  occurring  is  often  sufficient 
to  stop  the  activity  of  all  nitrogen-fixing  organisms,  if  not  to  kill 
them.  (4)  The  quantity  of  nitric  nitrogen  and  of  chlorin  in  any 
given  "  nitre  spot"  varies  in  the  same  spot  from  year  to  year  or  from 
period  to  period  within  a  year.  (5)  The  country  rock  adjacent  to 
the  nitrate  accumulations  and  which  has  contributed  to  the  soil 
formation  contains  abundance  of  nitrates  to  account  for  the  accumu- 
lations noted.  (6)  Soils  having  a  similar  physical  appearance  may 
be  produced  in  the  laboratory  in  the  absence  of  bacteria.  Because 
of  this,  we  must  conclude  that  the  accumulation  of  nitrates  in  spots 
in  western  soils  have  their  origin  as  do  other  accumulations  of 
soluble  salts  found  in  the  soil  and  not"  in  the  fixation  in  place  by 
bacterial  activity. 

Soil  Inoculation.— High  hope  was  entertained  that  the  nitrogen 
problem  in  agriculture  had  been  solved,  when  Caron  announced  that 
he  had  prepared  a  culture  of  bacteria  which  would  enable  non- 
leguminous  plants  to  utilize  free  atmospheric  nitrogen,  provided 
certain  precautions  were  observed.  Many  of  the  results  which  he 


SOIL  INOCULATION 


285 


reported  on  pot  experiments  were  clearly  in  favor  of  the  inoculated 
plant.  Stoklasa  was  one  of  the  first  to  study  in  detail  the  commercial 
preparation  "alinit"  which  was  placed  on  the  market  as  a  result  of 
^^aron's  work.  His  findings  were  fully  as  favorable  as  Caron's, 
jlBlthe  work  of  others  soon  demonstrated  that  "alinit"  neither  in 
the  laboratory  nor  in  the  field  had  the  ability  to  fix  nitrogen.  When 
Beijerinck  discovered  the  free-living  aerobic  nitrogen-fixers,  the  hope 
that  soil  inoculation  may  be  so  perfected  that  it  would  be  beneficial 
to  crops  was  revived,  and  since  that  time  many  investigators  have 
attempted  to  inoculate  soil  in  order  to  increase  its  crop-producing 
powers,  but  usually  with  negative  results.  Stoklasa  has  made 
great  claims  for  soil  inoculation.  He  found  that  soils,  inoculated 
with  Azotobacter  chroococcum  and  adequately  supplied  with  carbo- 
hydrates and  lime,  showed  an  increase  in  the  number  of  nitrogen- 
fixing  organisms,  and  also  an  increased  yield  both  in  quantity  and 
quality  of  the  crop.  Stranak  also  obtained  a  pronounced  increase 
in  the  production  of  beets,  grain,  and  potatoes  on  inoculating  with 
Azotobacter. 

There  may  be  a  decrease  in  the  crop  during  the  first  year  when 
carbohydrates  and  Azotobacter  are  added  to  the  soil  with  a  marked 
increase  in  crop  during  the  second  and  third  year.  Even  then,  the 
soil  may  be  left  richer  in  nitrogen  than  it  was  at  first. 

The  effect  of  dextrose  and  sucrose  on  the  productiveness  and  nitro- 
gen content  of  the  soil  is  shown  below: 


Crops  obtained. 

Total 

Total 

nitrogen 

Nitrogen 

Carbohydrate  added  per 
100  gms.  of  soil. 

Oats,  1905. 

Sugar  beets,  1906. 

nitrogen 
remained 
in  crop, 

left  in 
soil 
spring  of 

as 
nitrates, 
pts.  per 

Dry 
matter. 

Yield  of 
nitrogen. 

Dry 

matter. 

Yield  of 
nitrogen. 

gm. 

1906, 
per  cent. 

mil. 

None 

100  0 

100.0 

100.0 

100.0 

0.9514 

0.093 

10 

2  per  cent,  dextrose    . 

32.8 

62.5 

186.0 

190.0 

0.6814 

0.105 

17 

2  per  cent,  sucrose 

33.3 

58.7 

179.0 

195.0 

0.6800 

0.105 

15 

4  per  cent,  sucrose 

37.7 

78.1 

283.0 

339.0 

1.0092 

0.119 

37 

It  is  often  the  case  that  the  addition  of  starch  to  a  soil-during  the 
first  year  retards  plant  growth.  This  injurious  action  may  be  due 
to  the  augmented  bacterial  activity  in  the  soil  brought  about  by  the 
carbohydrates  which  injure  the  roots  of  the  plant  by  withdrawing 
oxygen  and  by  forming  hydrogen  sulphid  in  the  deoxygenated 
atmosphere  of  the  soil  through  the  reduction  of  sulphates  by  the 
bacteria. 

The  effect  produced  by  the  carbohydrate  applications  also  varies 
with  the  season.  If  applied  to  the  soil  in  the  spring  when  the  soil 
temperature  is  low  and  when  other  bacteria  are  more  active  than 


286  AZOFICATION 

Azotobacter,  the  results  are  that  they  rapidly  multiply  and  compete 
with  the  higher  plants  for  the  limited  available  plant-food.  If, 
however,  the  carbohydrates  are  applied  in  the  autumn  directly 
after  the  removal  of  the  crop,  when  the  soil  is  warm,  Azotobacter 
are  active,  with  the  result  that  sufficient  nitrogen  is  fixed  to  produce 
an  increased  crop  the  following  season. 

If  the  same  quantity  of  carbohydrates  per  unit  of  nitrogen  fixed 
be  required  by  the  organism  under  natural  conditions,  as  are  found 
necessary  in  laboratory  experiments,  enormous  quantities  would  be 
required  for  the  fixation  of  any  considerable  quantity  of  nitrogen; 
but  it  is  possible  that  in  the  soil  they  are  more  economical  with  their 
energy  or  they  may  live  in  symbiosis  with  other  organisms  which 
furnish  them  part  of  their  carbon. 

Many  workers  have  noted  either  no  effect  or  even  a  detrimental 
influence  when  soils  are  treated  with  the  carbohydrates  and  then 
inoculated  with  Azotobacter.  This  may  be  due  in  a  great  measure  to 
any  or  all  of  the  following  factors:  (a)  Absence  of  a  suitable 
environment,  as  temperature,  moisture,  aeration,  food  and  alkalinity; 
(6)  absence  of  a  suitable  host  from  which  Azotobacter  may  obtain 
part  of  its  carbon;  (c)  injurious  effects  due  to  the  decomposition 
products  of  the  carbohydrate  added. 

There  is  considerable  interest  in  the  work  of  Bottomley  who 
uses  bacterized  peat,  or  humogen.  The  bacterizing  process  consists 
of  three  stages :  (a)  Treatment  of  peat  with  a  culture  solution  of  the 
special  "humating"  bacteria  and  an  incubation  of  it  at  constant 
temperature  for  a  week  or  ten  days,  during  which  period  soluble 
humates  are  formed;  (6)  destruction  of  the  humating  bacteria  by 
sterilization  with  live  steam;  (c)  treatment  of  this  sterilized  peat 
with  mixed  cultures  of  nitrogen-fixing  organisms— Azotobacter 
chroococcum  and  Bacillus  radicicola—smd  an  incubation  at  20°  C.  for 
a  few  days,  after  which  it  is  ready  for  use. 

Theoretically,  there  is  much  in  this  process  which  recommends  it, 
for  there  is  no  abrupt  change  in  environmental  conditions  for  the 
organism  added,  as  would  be  the  case  when  added  from  laboratory 
culture.  Moreover,  they  are  added  in  enormous  quantities  and 
with  a  source  of  carbon  which  is  not  far  different  from  that  found  in 
the  soil.  Russell,  however,  after  carefully  reviewing  all  of  the 
experimental  evidence  on  the  subject,  concludes:  "There  is  no 
evidence  that  humogen  possesses  any  special  agricultural  value. 
There  is  not  the  least  indication  that  it  is  fifty  times  as  effective  as 
farmyard  manure,  to  quote  an  often  repeated  statement,  and  there 
is  nothing  to  show  that  it  is  any  better  than  any  other  organic 
manure  with  the  same  nitrogen  content."  Furthermore,  he  con- 
cludes that  there  is  no  definite  evidence  that  "  bacterization"  really 
adds  to  the  value  of  peat. 

The  conclusion  is  evident  that  soil  inoculation,  in  order  to  be 


SOIL  INOCULATION  287 

successful,  must  be  accom'panied  by  the  rendering  of  the  physical 
and  chemical  properties  of  the  soil  ideal  for  the  growth  of  the  specific 
organisms  to  be  added.  A  few  organisms  placed  in  a  new  environ- 
ment already  containing  millions  can  never  hope  to  gain  the  ascend- 
ency over  the  organisms  naturally  occurring  in  the  soil,  for  they  have 
been  struggling  for  countless  generations  to  adapt  themselves  to  the 
environment  and  only  those  which  are  fitted  have  survived.  The 
problem  becomes  even  more  complicated  when  we  recall  the  findings 
of  Lipman  that  the  bacterial  flora  of  a  soil  is  in  many  cases  entirely 
changed  by  climatic  conditions.  On  this  account,  it  would  appear 
that  ever  to  make  soil  inoculation  a  success  the  chemical,  physical, 
and  even  the  biological  condition  must  be  made  suitable  for  the 
growth  of  the  specific  organism  added.  Furthermore,  strains  of  the 
organism  must  be  used  which  have  been  evolved  under  similar 
climatic  conditions. 

Soil  Gains  in  Nitrogen.— It  is  well  established  that  many  forms  of 
microscopic  organisms  possess  the  power  of  fixing  nitrogen  either 
when  grown  alone  or  in  combination  with  other  organisms  of  the 
soil.  Many  of  these  have  been  obtained  in  pure  culture  and  their 
morphology  and  physiology  carefully  studied.  The  most  favorable 
conditions  for  their  maximum  nitrogen-fixation  in  pure  cultures  in 
liquid  solutions  have  been  accurately  determined.  Some  of  the 
conditions  requisite  for  their  activity  in  soils  are  known,  but  on  this 
phase  of  the  subject  there  are  many  gaps  in  our  knowledge  and  much 
work  must  yet  be  done  before  we  can  state  definitely  the  part  which 
they  play  in  the  economy  of  nature  and  before  we  can  say  which 
are  the  very  best  methods  for  increasing  their  usefulness.  Never- 
theless, it  is  interesting  to  consider  the  results  obtained  by  a  few 
workers. 

Berthelot's  early  laboratory  experiments  led  him  to  believe  that 
sands  and  clays  may  fix  in  a  year  from  75  to  100  pounds  of  nitrogen 
to  the  acre.  In  two  exceptional  instances  he  noted  that  nitrogen 
was  fixed  by  sands  at  the  rate  of  525  pounds  and  980  pounds  an  acre, 
but  soils  which  contained  fairly  large  quantities  of  nitrogen  never 
made  markedly  rapid  gains. 

Thiele,  on  the  other  hand,  maintained  that  while  there  is  no  doubt 
that  Azotobacter  possessed  the  power  of  fixing  freenitpegen,  under 
laboratory  conditions,  yet  it  is  not  certain  that  conditions  would  be 
such  in  soils  for  any  gain  of  nitrogen  due  to  the  activity  of  these 
organisms.  We  have  already  seen,  however,  that  the  Azotobacter 
do  not  require  as  high  a  temperature  for  nitrogen-fixation  in  soil  as 
he  thought  necessary.  It  is  also  certain  that  in  most  arable  soil  the 
temperature  is  sufficient  during  a  large  part  of  the  year  for  a  fairly 
rapid  nitrogen-fixation  by  bacteria. 

Krainsky  thinks  that  even  better  results  should  be  obtained  in 
soils  than  in  pure  culture,  for  there  the  nitrogen-fixers  grow  in  sym- 
biosis with  autotrophic  organisms  which  make  organic  compounds 


288  AZOFICATION 

available  to  the  Azotobacter.  In  soils  the  nitrogen  fixed  is  rapidly 
removed  by  other  plants,  because  of  which  the  slowing-up  process 
that  becomes  perceptible  so  early  in  laboratory  experiments  should 
not  occur. 

In  addition  to  an  optimum  temperature  and  moisture  content  of 
the  soil,  the  Azotobacter  are  dependent  upon  a  supply  of  carbon  for 
energy  and  inorganic  nutrients  for  the  building  of  cell  protoplasm. 
Unfortunately,  it  is  too  often  the  case  that  under  natural  conditions 
those  soils  which  are  deficient  in  nitrogen  are  also  lacking  in  available 
carbon,  and  especially  in  phosphorus,  which  are  so  essential  for  rapid 
nitrogen-fixation.  Then  there  are  the  technical  difficulties  which 
the  chemist  encounters  in  determining  the  gain  or  loss  of  nitrogen 
which  occurs  in  soils  under  natural  conditions  and  which  may  be 
attributed  to  non-symbiotic  nitrogen-fixation. 

There  are,  however,  several  cases  in  which  the  gain  has  been 
measured  with  a  fair  degree  of  accuracy. 

Lipman,  in  pot  experiments  carried  on  with  a  soil  containing  about 
5000  pounds  of  nitrogen  per  acre-foot  of  soil,  found  again  of  more 
than  one-third  this  amount  in  two  short  seasons^  Much  of  this 
must  be  attributed  to  non-symbiotic  nitrogen-fixation.  To  these 
soils  had  been  applied  solid  and  liquid  manure;  which  furnished  to 
the  organisms  readily-available  supplies  of  energy  and  various 
necessary  inorganic  constituents.  This  fixation  was  not  nearly  so 
rapid  where  legumes  were  turned  under  as  green  manures. 

Koch  found  a  gain  of  from  0.019  to  0.093  per  cent,  in  soil  nitrogen 
during  two  seasons  which  must  be  attributed  to  non-symbiotic 
nitrogen-fixation.  In  addition  to  this  there  was  a  threefold  gain  in 
the  nitrogen  content  of  the  crops— oats,  buckwheat,  and  sugar-beets 
—which  must  also  be  attributed  to  the  action  of  Azotobacter. 

Hall  noted  an  annual  gain  of  100  pounds  of  nitrogen  on  Broadbalk 
field  at  Rothamsted  and  25  pounds  on  Grescroft  field.  He  feels  that 
much  of  this  gain  must  be  due  to  the  action  of  non-symbiotic 
bacteria.  Lipman  points  out  that  the  actual  gains  of  nitrogen  are 
even  greater,  for  this  does  not  take  into  consideration  the  various 
losses  which  are  sure  to  occur  even  under  the  best  of  conditions. 
Hopkins  takes  the  stand  that  the  apparent  gain  is  due  in  a  large 
measure  to  drifting  dust  and  plant  residues  coupled  with  the  diffi- 
culty of  obtaining  representative  samples  of  soil  at  the  two  different 
periods.  Even  when  all  of  these  factors  are  considered  the  evidence 
points  to  a  gain  of  nitrogen  through  bacterial  activity. 

The  analysis  of  a  great  number  of  soils  in  Utah  showed  that  the 
average  nitrogen  content  of  the  soil  which  had  grown  wheat  and 
other  non-leguminous  plants  for  from  twenty  to  fifty  years  was 
0.2009  per  cent.,  whereas  adjoining  virgin  soil  on  the  average  showed 
only  0.1984  per  cent,  of  total  nitrogen.  *The  evidence  is  very  strong 
that  considerable  nitrogen,  fes  been  added  to  these  soils  by  micro- 
scopic organisms,  for: 


SOIL  INOCULATION  289 

(a)  In  nearly  every  case  the  cultivated  soil  fixed  much  more 
nitrogen  in  the  laboratory  than  did  the  virgin  soil.     This  was  the 
case  when  the  soil  was  incubated  with  or  without  the  addition  of 
carbonaceous  material. 

(b)  There  is  a  richer  nitrogen-fixing  bacterial  flora  in  the  cultivated 
than  in  the  virgin  soil. 

(c)  The  conditions  of  moisture,  alkalinity  and  food  constituents 
in  the  soil  were  ideal  for  rapid  nitrogen-fixation,  and  the  temperature 
of  the  soil  was  high  enough  during  a  considerable  part  of  the  year 
for  the  growth  of  Azotobacter. 

(d)  The  cultivation  of  the  soil  would  increase  aeration  and  avail- 
able phosphorus  in  the  soil. 

(e)  The  large  quantity  of  plant  residues  would  act  as  a  supply  of 
carbon  which  is  readily  rendered  available  by  the  soil's  rich  flora  of 
cellulose  ferments.     If  these  soils  had  produced  a  wheat  crop  every 
alternate  year  and  all  of  the  nitrogen  which  had  been  added  to  the 
soil  without  loss  from  leaching  or  bacterial  activity  taken  by  the 
crop,  it  would  have  necessitated  the  addition  of  25  pounds  an  acre 
yearly,  which  is  evidently  the  very  minimum  which  can  be  attributed 
in  these  soils  to  non-symbiotic  nitrogen-fixation. / 

Eighty  different  samples  of  these  soils  were  incubated  in  the 
laboratory  for  twenty-one  days  and  the  gains  in  nitrogen  determined 
by  comparing  with  sterile  checks.  The  soils  were  incubated  without 
the  addition  of  anything  except  sterile  distilled  water.  At  the  end 
of  the  period  the  average  gain  per  acre  for  the  cultivated  soils  was 
202  pounds  and  that  for  the  virgin  soil  was  92. 

True,  fixation  would  not  continue  long  at  this  rate,  for  when  the 
nitrogen  content  of  the  soil  passed  beyond  a  certain  limit  decay 
bacteria  would  increase  rapidly,  and  in  the  struggle  for  existence 
they  are  able,  with  the  advantage  at  their  disposal,  to  suppress  the 
more  slowly  growing  Azotobacter,  which  would  gain  the  ascendency 
again  only  when  the  nitrogen  of  the  soil  became  low. 

Thus,  there  is  an  upper  as  well  as  a  lower  limit  to  the  nitrogen 
content  of  the  soil  as  far  as  bacterial  activity  is  concerned,  but  by 
making  the  conditions  for  nitrogen-fixation  as  nearly  ideal  as  possible 
we  may  maintain  in  a  soil  the  upper  and  not  the  lower  nitrogen 
content. 

^In  conclusion,  it  may  be  stated  that  although  the  part  played  by 
Azotobacter  in  maintaining  the  nitrogen  of  the  soil  has  not  been 
definitely  measured,  it  is  nevertheless  an  important  factor.  Hall 
found  it  to  be  at  least  25  pounds,  Lohnis  35.7  pounds,  and  the  author 
25  pounds  per  acre  annually.  It  is,  therefore,  conservative  to  state, 
as  has  Lipman,  that  these  organisms,  under  favorable  conditions, 
add  from  15  to  40  pounds  of  available  nitrogen  to  each  acre  of  soil 
yearly. 

REFERENCE. 

Greaves,  J.  E.:     Azofication,  Soil  Science,  1918,  vi,  163-217. 
19 


CHAPTER   XXIV. 
SYMBIOTIC  NITROGEN  FIXATION. 

FROM  the  earliest  day  of  agricultural  practice  it  has  been  the 
experience  of  practical  men  that  legumes  under  appropriate  condi- 
tions render  the  soil  more  productive.  It  was  the  practice  of  the 
Roman  farmers  to  plow  under  lupines  in  order  to  enrich  their  soil. 
This  practice  has  persisted  through  all  the  succeeding  ages  by  the 
farmers  of  Europe  and  Asia.  But  it  is  only  within  the  memory  of 
men  now  living  that  we  have  been  able  to  state  the  cause  of  the 
increased  fertility. 

Early  Theories.— Liebig,  by  applying  the  exact  methods  of  chemis- 
try to  agriculture,  was  able  to  demonstrate  that  plants  get  their 
carbon  from  the  carbon  dioxid  of  the  air  and  not  from  the  carbon 
compounds  of  the  soil.  He  came  to  regard  the  ammonia  of  the  air 
as  analogous  to  the  carbon  dioxid  and  taught  the  doctrine  that  the 
plants  are  able  to  derive  their  nitrogenous  food  from  the  atmosphere. 
He  wrote:  "If  the  soil  be  suitable,  if  it  contains  a  sufficient  quan- 
tity of  alkalies,  phosphates,  and  sulphates,  nothing  will  be  wanting. 
The  plants  will  derive  their  ammonia  from  the  atmosphere  as  they 
do  carbonic  acid."  Liebig  considered  all  crops  capable  of  securing 
the  nitrogen  from  the  air,  but  the  legumes  and  other  broad-leafed 
plants  were  especially  fitted  for  this  task,  as  is  witnessed  by  the 
fact  that  they  benefit  the  succeeding  cereal  crops  and  do  not  respond 
as  readily  to  nitrogenous  fertilizers. 

It  was  soon  proved  that  the  ammonia  and  other  nitrogen  com- 
pounds of  the  air  which  were  brought  down  by  snow  and  rain  were 
very  small  and  would  account  for  only  a  small  fraction  of  the  nitro- 
gen removed  by  the  crops. 

Lawes  and  Gilbert  (1855)  reached  the  conclusion  that  non- 
leguminous  plants  require  a  supply  of  some  nitrogenous  compound, 
nitrates  and  ammonium  salts  being  about  equally  effective.  The 
amount  of  ammonia  obtainable  from  the  atmosphere  is  insufficient 
for  the  need  of  crops.  Leguminous  plants  behave  abnormally. 

They  took  the  precaution  of  calcining  the  soil  and  removing  all 
of  the  ammonia  from  the  air  before  it  was  admitted  to  the  vessel  in 
which  the  plants  were  grown.  Their  results  and  those  of  Boussin- 
gault  agree  fully  in  pointing  to  the  conclusion  that  free  nitrogen  of 
the  air  was  not  available  to  the  plants.  These  conclusions  were 
accepted  as  decisive  for  a  number  of  years,  although  much  evidence 


EARLY  OBSERVATIONS  ON  ROOT  TUBERCLES          291 

pointed  in  the  other  direction.  Pot  and  field  experiments  carried 
out  in  England,  France,  Germany,  and  the  United  States  during  the 
early  eighties  furnished  unmistakable  evidence  that  the  legumes 
possessed  the  power  of  utilizing  atmospheric  nitrogen.  Atwater's 
experiments  (1883-84)  fully  demonstrated  this.  In  some  of  his 
trials  the  nitrogen  gained  was  50  per  cent,  or  more  of  the  total 
quantity  harvested.  However,  the  mystery  was  not  solved  until 
1886  when  Hellriegel  and  Wilfarth  announced  that  the  fixation  of 
free  nitrogen  is  a  property  possessed  by  the  legumes  and  is  due  to 
the  bacteria  associated  with  them  in  the  root  tubercles. 

Early  Observations  on  Root  Tubercles.— The  presence  of  tubercle 
on  the  roots  of  leguminous  plants  had  long  before  been  noted  by 
Malpighi.  He  regarded  them  a's  root  galls.  Later  they  were 
regarded  as  buds  of  incomplete  plants,  or  as  rudimentary  roots. 
In  1866  Woronin  found  in  them  numerous  minute  bodies  which  bore 
some  resemblance  to  bacteria.  They  were  rod-shaped  but  often 
slightly  forked  to  "T"-  or  "Y"-shaped  bodies.  On  account  of  this 
irregularity  in  shape  the  discoverer  was  unable  to  say  whether  they 
were  true  bacteria  or  not.  He,  therefore,  called  them  bacteroids, 
and  regarded  them  as  the  cause  of  the  tubercles.  In  1874  Erickson 
found  that  in  the  early  stages  of  the  development  of  the  tubercle  it 
was  filled  with  long,  branching  threads  resembling  the  mycelium  of 
fungi,  and  to  these  hyphse  he  attributed  the  formation  of  the  tuber- 
cles. In  later  stages  of  the  growth  of  the  tubercles  he  found  bac- 
teroids, but  was  unable  to  determine  whether  they  had  any  connec- 
tion with  the  hyphse  or  not. 

Frank  (1879)  not  only  showed  that  tubercles  are  almost  invariably 
present  on  the  roots  of  legumes  but  that  their  formation  may  be 
prevented  by  the  sterilization  of  the  soil.  He  was  thus  in  possession 
of  facts  which  might  have  revealed  to  him  the  true  nature  of  the  root 
tubercles.  However,  he  accepted  the  interpretation  of  his  pupil, 
Brunchhorst,  who  claimed  the  bacteria-like  bodies,  were  merely 
reserve  food  materials. 

Marshall  Ward  not  only  proved  that  tubercle  formation  is  due  to 
outside  infection  but  that  such  infection  may  be  brought  about  by 
placing  pieces  of  old  tubercles  in  contact  with  the  roots  of  growing 
leguminous  plants. 

Hellriegel  found,  as  the  result  of  a  long  series  of  experiments,  that 
when  pea  plants  were  grown  in  sterilized  soils  as  a  rule  no  tubercles 
were  formed,  but  when  the  plants  were  watered  with  soil  infusions 
made  by  allowing  water  to  act  upon  soil  in  which  peas  had  been 
grown,  the  tubercles  appeared  in  abundance.  If  the  soil  infusion 
was  sterilized  by  boiling  before  it  was  put  upon  the  plants  no  tuber- 
cles appeared.  These  experiments  were  thought  to  prove  that  the 
tubercles  were  really  caused  by  living  organisms  in  the  soil  infusion, 
which  were  killed  by  heat.  The  tubercles  could  not,  therefore,  be 


292  SYMBIOTIC  NITROGEN  FIXATION 

regarded  as  normal  products  of  the  roots,  but  were  certainly  infec- 
tions from  the  soil.  In  a  series  of  researches,  undertaken  with  the 
assistance  of  Wilfarth  these  results  were  thoroughly  confirmed. 
They  showed  that  in  sterilized  soil  the  legume  behaves  the  same  as 
the  non-legume  and  dies  of  nitrogen  hunger  if  not  supplied  with 
suitable  forms  of  nitrogen.  When  the  sterilized  soil  was  inoculated 
with  fresh  soil  on  which  legumes  had  made  a  normal  growth  they 
then  made  a  vigorous  growth  in  sterilized  soil.  Under  similar 
conditions  non-legumes  did  not  recover.  The  recovery  of  the 
starving  legume  was  found  to  coincide  with  the  formation  of  root 
tubercles. 

Wigand  (1887)  found  that  the  tubercles  contained  true  bacteria 
and  the  following  year  these  were  obtained  in  pure  cultures  by 
Beijerinck.  He  found  further  that  there  were  bacteria  associated 
with  all  tubercles,  and  although  the  bacteria  differed  somewhat  in 
the  tubercles  of  different  species  of  plants,  still  there  were  certain 
constant  characteristics  to  be  seen  in  them  all.  He,  therefore, 
regarded  the  tubercles  as  the  result  of  the  action  of  bacteria  and  gave 
to  the  organism  producing  the  tubercles  the  name  of  Bacillus 
radicicola.  Beijerinck  regarded  the  so-called  bacteroids  of  Woronin 
as  degenerate  forms  of  the  bacteria-involution  forms,  which  appeared 
only  after  the  bacteria  had  lost  their  vigor.  In  a  later  investigation , 
after  isolating  the  bacteria  and  keeping  them  in  pure  cultures  for 
many  months,  he  was  able  to  produce  the  tubercles  at  will  by  inocu- 
lating soils  in  which  his  plants  were  grown  with  the  pure  cultures  of 
the  organisms. 

Prazmowski  (1890)  published  researches  which  confirmed  all  of 
HellriegePs  results,  showing  conclusively  that  if  sufficient  precau- 
tions were  taken  to  sterilize  the  soil  in  which  leguminous  plants  were 
grown  no  tubercles  were  ever  produced.  He  further  showed  that 
the  tubercles  grow  on  plants  developing  both  in  the  light  and  in  the 
dark,  but  are  larger  on  plants  growing  in  the  light;  that  they  only 
appear  on  healthy  plants;  that  there  are  very  few  on  plants  growing 
in  well-washed  sand;  that  if  plants  growing  in  sterilized  soil  be 
watered  with  brook  or  river  water,  tubercles  occasionally  develop 
but  never  in  abundance;  and  that  the  infection  of  the  roots  occurs 
early  in  the  germination  of  the  plant  and  cannot  take  place  in  the 
older  roots. 

Two  years  later  Schlosing  and  Laurent  demonstrated  the  fixation 
of  atmospheric  nitrogen  through  the  joint  activities  of  leguminous 
plants  and  Pseudomonas  radicicola  by  the  actual  diminution  of  the 
amount  of  elementary  nitrogen  in  the  inclosed  atmosphere  sur- 
rounding the  plants. 

Species.— Whether  the  different  varieties  of  legume  bacteria  are 
distinct  species  is  a  perplexing  question  which  today  cannot  be 
definitely  answered.  It  is  known  that  certain  legumes  are  readily 


SPECIES  293 

infected  by  one  variety,  whereas  with  another  variety — infection  is 
accomplished  with  difficulty  or  not  at  all.  Moreover,  the  sero- 
logical  test  yielded  by  different  varieties  is  specific.  These  facts  have 
led  some  observers  to  consider  the  types  as  quite  distinct,  whereas 
others  consider  them  as  simply  physiological  varieties  of  the  same 
general  species.  On  the  whole,  the  consensus  of  opinion  at  the 
present  time  seems  to  be  decidedly  in  favor  of  this  latter  view. 

Some  of  Hellriegel's  experiments  indicated  that  bacteria  from 
clover  could  not  produce  tubercles  on  lupines  and  serradella.  Simi- 
lar results  were  obtained  by  Nobbe  and  his  associates,  yet  they  were 
finally  led  to  conclude  that  the  root  invasion  of  legumes  is  caused  by 
a  single  species.  Long-continued  growth  of  the  organism  on  a 
legume  adapts  it  to  that  legume  so  it  no  longer  invades  the  roots  of 
other  legumes.  But  Petermann  '(1893)  considered  it  probable  that 
every  genus  of  plant  has  its  specific  bacteria.  Buhlert  considered 
that  all  of  the  organisms  are  forms  of  B.  radicicola  but  that  the 
bacteria  best  adapted  to  a  given  species  of  leguminous  plant  are  those 
naturally  found  upon  that  plant.  However,  cross  inoculation  is 
possible  within  certain  limits.  From  the  root  tubercles  of  some 
leguminous  plants  he  obtained  bacteria  which  seemed  to  be  very 
highly  specialized,  but  he  considers  that  this  specialization  does  not 
extend  to  differences  that  may  be  regarded  as  specific. 

As  a  result  of  a  large  number  of  experiments  with  different  kinds 
of  legumes,  Maassen  and  Miiller  (1907)  reached  the  conclusion  that: 
(1)  The  organisms  of  Pisum  satium  will  inoculate  Vicia  faba,  V. 
saliva,  V.  villosa,  Lens  esculenta,  Laihyrus  sativus,  L.  odoralus, 
and  L.  silvestris;  (2)  that  of  Trifolium  incarnatum  will  inoculate 
T.  pratense;  (3)  that  of  Medicago  saliva  will  inoculate  M.  lupulina 
and  Melilotus  officinalis;  and  (4)  that  of  Lupinus  lutens  will  inoculate 
L.  angustifolius  and  Ornilhopus  salivus.  The  organisms  of  Phaseolus 
vulgaris,  Soja  hispida,  and  Robinia  pseudacacia,  according  to 
Maassen  and  Miiller,  will  apparently  not  inoculate  any  other  plant. 
Similar  conclusions  were  reached  regarding  the  organisms  of  Coronilla 
varia,  Onobrychis  satava,  Anthyllis  vulneraria,  Sarclhamnus  scoparius, 
Amorpha  frnticosa,  Caragana  frutescens,  and  Acacia  lophanta. 

De  Rossi  (1907)  described  a  specific  organism  derived  by  him  from 
root  tubercles  of  V.  faba  which  produces  root  tubercles  and  which 
he  claimed  is  morphologically,  biologically,  and  culturally  widely 
different  from  Bacillus  radicicola  Beijerinck. 

Nobbe  and  coworkers  (1908)  showed  that  pure-  cultures  of 
bacteria  from  tubercles  of  one  member  of  a  genus  are  effective  on 
other  members  of  the  same,  and,  as  a  rule,  only  of  the  same  genus. 
They  found,  however,  complete  interchangeability  in  case  of  peas 
and  vetches  and  partial  in  case  of  lupines  and  serradella. 

Zipfel  (1912),  with  the  hope  of  throwing  light  upon  the  kinship 
among  the  various  nodule  bacteria,  made  use  of  .the  agglutination 


294 


SYMBIOTIC  NITROGEN  FIXATION 


II 
1 1 


0 

1 


SPECIES  295 

method.  From  his  results  he  concluded  that  the  nodule  bacteria 
were  not  varieties  of  the  same  species,  but  that  distinct  species 
existed. 

Klimmer  and  Kriiger  two  years  later  used  serological  tests  to 
distinguish  species.  They  used  the  agglutination  method  princi- 
pally and  complement  binding  and  precipitation  for  confirmation  and 
control.  Working  with  organisms  from  eighteen  legume  species, 
they  divided  the  bacteria,  according  to  their  methods,  into  nine 
species  which  they  asserted  differed  sharply  from  one  another. 

Simon  (1914)  tested  various  cultures  upon  seedlings  of  several 
legume  species  and  compared  the  results  with  those  obtained  by 
using  Zipfel's  agglutination  method.  He  found  that  the  results  of 
both  methods  agree  substantially.  His  grouping  of  the  organisms 
is  in  general  agreement  with  that  of  Klimmer  and  Kriiger.  He 
concluded,  however,  that  "the  root  bacteria  of  legumes  are  rather 
to  be  conceived  as  more  or  less  constant  adaptations  of  the  species 
Bacillus  radicicola." 

Burrill  and  Hansen  (1917),  after  an  extensive  study  of  various 
legume  bacteria  both  with  the  pot-culture  method  and  the  agar 
test-tube  method  of  Garman,  divided  the  nodule  organisms  into  the 
following  eleven  groups  according  as  they  are  interchangeable  for 
the  purpose  of  inoculation: 

GROUP  I. 

Mammoth  red  clover,  Trifolium  pratense  perenne. 

Alsike,  or  Swedish  clover,  Trifolium  hybridum. 

Crimson  clover,  Trifolium  incarnatum. 

Berseem,  or  Egyptian  clover,  Trifolium  alexandrianum. 

White  clover,  Trifolium  repens. 

Zigzag,  or  cow  clover,  Trifolium  medium. 

GROUP  II. 

White  sweet  clover,  Melilotus  alba. 

Yellow  sweet  clover,  Melilotus  officinalis. 

Wild  yellow  sweet  clover,  Melilotus  indica. 

Alfalfa,  Medicago  sativa. 

Alfalfa,  Medicago  falcata. 

Bur  clover,  Medicago  hispida. 

Black  medick,  or  yellow  trefoil,  Medicago  lupalina. 

Funugreek,  Trigonella  foenum-graecum. 

GROUP  III. 

Cowpea,  Vigna  sinensis. 
Partridge  pea,  Cassica  chamaecrista. 
Peanut,  Archis  hypogoea. 
Japan  clover,  Lespedeza  striata. 


296  SYMBIOTIC  NITROGEN  FIXATION 

Slender  bush  clover,  Lespedeza  virginica. 
Velvet  bean,  Mucuna  utilis. 
Wild  indigo,  Baptisia  tinctoria. 
Tick  trefoil,  Desmodium  canescens. 
Tick  trefoil,  Desmodium  illinoense. 
Acacia,  Acacia  armata. 
Acacia;  Acacia  floribunda. 
Acacia,  Acacia  longifolia. 
Acacia,^4cocm  melanoxylon. 
Acacia,  Acacia  semperflora. 
Acacia,  Acacia  from  California. 
Dyer's  greenweed,  Genista  tinctoria. 

GROUP  IV. 

Common  garden  pea,  Pipsum  sativum. 

Field  pea,  or  Canada  field  pea,  Pisum  sativum  arvense. 

Hairy  vetch,  Vicia  mllosa. 

Spring  vetch,  Vicia  sativa. 

Broad  bean,  Vicia  faba. 

Narrow-leaved  vetch,  Vicia  angustifolia. 

Vetch,  Vicia  daysiecarpa. 

Lentil,  Lens  escuknta. 

Sweet  pea,  Lathyrus  odoratus. 

Perennial  pea,  Lathyrus  latifolius. 

GROUP  V. 
Soybean,  Glycine  hispida. 

GROUP  VI. 

Garden  bean,  Phaseolus  vulgaris. 
Garden  bean,  Phaseolus  angustifolia. 
Scarlet  runner  bean,  Phaseolus  multiflorus. 

GROUP  VII. 

Lupine,  Lupinus  perennis. 
Serradella,  Ornithopus  sativus. 

GROUP  VIII. 
Hog  peanut,  Amphicarpa  monoica. 

GROUP  IX. 
Lead  plant,  Amorpha  canescens. 


CULTURAL  CHARACTERISTICS  297 

GROUP  X. 
Trailing  wild  bean,  Strophostyles  helvola. 

GROUP  XL 
Black,  or  common  locust,  Robinia  pseudo-acacia. 

Hiltner  and  Stormer  (1903),  however,  arranged  the  tubercle 
bacteria  into  two  groups  possessing,  according  to  them,  well-defined 
morphological  and  physiological  differences.  One  of  these  groups 
is  included  under  the  species  Rhiz^b^um  radicicola  and  the  other 
under  Rhizobium  beijerinckii.  The  former  comprises  the  organisms 
from  lupines,  serradella,  and  soybeans,  whereas  the  latter  comprises 
all  of  the  others. 

Grieg-Smith  (1902)  reports  having  found  three  races  of  the 
nodule  bacteria  in  the  same  nodule,  while  Gino  de  Rossi  (1907) 
reported  the  finding  of  two  organisms  which  differ  in  that  one  forms 
a  large  hyaline  colony  not  developing  well  on  beef  and  peptone 
gelatin,  while  the  other  forms  white  non-transparent  colonies  on  beef 
gelatin.  He  believes  that  the  one  is  morphologically,  biologically, 
and  culturally  widely  different  from  Bacillus  radicicola  (Beijerinck). 

Cultural  Characteristics.— The  nodule  bacteria  grow  well  on  a  great 
variety  of  cultural  media,  perhaps  best  on  a  medium  of  ash-maltose- 
agar  or  one  of  legume  extract  to  which  has  been  added  a  sugar, 
dextrose,  sucrose,  or  maltose,  and  dipotassium  phosphate. 

In  an  agar  stab  typical  drop-form  colonies  are  produced  at  the 
surface,  while  a  thin  gray  growth  follows  the  line  of  the  needle.  In 
standard  beef  broth  the  growth  of  the  organism  is  slow.  The  liquid 
becomes  cloudy,  a  gray-white  ring  is  formed,  and  a  thin  membrane 
covers  the  surface.  Later  a  flocculent  precipitate  settles  to  the 
bottom  of  the  tube.  In  standard  beef  broth  gelatin  the  growth  of  the 
organism  is  at  first  funnel-shaped  and  then  stratiform.  Gelatin  is 
slowly  liquefied,  the  process  sometimes  requiring  two  or  three  months 
for  completion.  In  gelatin  stabs  the  growth  sometimes  seals  over 
the  stab  with  a  drop-form  growth  and  liquefaction  does  not  occur. 
On  the  ordinary  cultural  media  the  organisms  do  not  show  any  very 
characteristic  growth.  The  most  noticeable  difference  between 
various  strains  is  the  rapidity  of  development.  Slight  alkalinity  to 
+20°  to  +25°  acid  (Fuller's  scale)  with  phenolphthalein  is  tolerated; 
neutral  to  +  10°  is  best. 

The  results  obtained  by  Fred  and  Davenport  clearly  indicated 
that  the  nodule  bacteria  from  different  plants  behave  differently 
toward  acid.  They  divided  the  legume  bacteria  into  five  groups 
depending  upon  their  sensitiveness  to  acid. 

1.  Critical  pn  4.9 Alfalfa  and  sweet  clover. 

2.  Critical  pn  4.7 Garden  pea,  field  pea  and  vetch. 

3.  Critical  pn  4.2 Red  clover  and  common  beans. 

4.  Critical  pn  3.3 Soybeans  and  velvet  beans. 

5.  Critical  pn  3.15 Lupines. 


298 


SYMBIOTIC  NITROGEN  FIXATION 


The  alfalfa  organism  is  the  most  sensitive  of  the  legume  bacteria 
to  acidity  and  conversely  the  lupine  organism  is  the  most  resistant  to 
acidity. 


FIG.  36 


FIG. 


FIGS.  36  and  37. — Ash-agar  plate  from  bean  (Phaseolus  vulgaris),  showing  giant 
colonies  in  a  thickly  seated  plate.  Ash-agar  plate  from  perennial  pea  (Dathyrus 
latifolius) ;  the  clear  spaces  are  due  to  sterilized  fluid  carried  over  with  pieces  of 
nodule  tissue.  (After  Burrill  and  Hansen). 


MORPHOLOGY  OF  THE  COLONIES 


299 


The  zone  of  optimum  temperature  (Zipfel)  is  between  18°  and  20° 
C.;  the  limits  of  growth  are  3°  and  45°  C.;  and  the  upper  limits  of 
life  are  from  60°  to  62°  C.  Burrill  gives  a  considerably  higher 
optimum  temperature— 25°  to  28°  C.  Grieg-Smith  found  the  best 
temperature  for  the  production  of  slime  to  be  22°  C.  with  most  organ- 
isms and  26°  C.  for  one  obtained  from  Robinia.  The  organism  is 
aerobic,  and  he  found  that  diffused  sunlight  of  the  laboratory  is  not 
harmful;  even  exposure  to  direct  sunlight  for  several  months  with- 
out transferring  did  not  k'ill  the  organisms  when  grown  upon  favor- 
able media  with  precautions  to  prevent  evaporation. 

Ball  found  that  the  organism  endures  at  least  two  years  in  dry 
soil.  Harrison  and  Barlow  found  that  the  limit  of  viability  on  ash- 
maltose-agar  varied  somewhat,  but  in  the  majority  of  cases  it  was 
about  two  years.  How  long  the  organism  will  exist  in  a  soil  under 
field  conditions  is  not  yet  known,  but  practical  observations  indicate 
that  it  must  be  many  years. 


FIG.  38. — Young  nodule  magnified,  showing  affected  root  hair  and  same  root  hair 
more  highly  magnified.     (After  Atkinson.) 

Morphology  of  the  Colonies.— Two  types  of  colonies  appear  on  agar 
plates— buried  and  surface  colonies— and  are  thus  described  by 
Burrill  and  Hansen: 


300 


SYMBIOTIC  NITROGEN  FIXATION 


"Buried  colonies  are  small  and  submerged,  most  frequently  lens, 
or  spindle-shaped,  with  smooth  and  even  edges.  They  are  rather 
opaque,  granular  in  structure,  and  in  color  are  cream  to  a  chalk 
white.  They  increase  slowly  in  size,  eventually  appearing  on  the 


FIG.  39. — Young  nodule,  showing  the  beginning  of  the  differentiation  of  its  tissues. 

(After  Prazmowski.) 

surface  of  the  agar  as  surface  colonies,  when  the  growth  becomes 
rapid.  The  lens  colonies,  however,  remain  visible  for  many  days  in 
the  center  of  the  new  growth. 

"Surface  colonies  originate  at  or  near  the  surfaces  of  the  agar  or 
develop  from  buried  colonies.    They  are  drop-form,  watery,  muci- 


MORPHOLOGY  OF  THE  COLONIES 

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302  SYMBIOTIC  NITROGEN  FIXATION 

laginous  (in  appearance,  though  not  always  to  the  touch),  gray- 
white  to  pearly  white  in  color,  glistening,  and  semitranslucent 
to  opaque.  The  edges  are  smooth  and  even.  Under  the  low  power 
the  interior  is  granular.  They  frequently  attain  considerable  size, 
a  centimeter  or  more  in  diameter. 

"  Plates  made  direct  from  the  nodule  lack  uniformity  to  a  marked 
degree.  The  undiluted  plate  (first  plate)  begins  to  show  a  few 
colonies  in  two  to  four  days.  These  colonies  become  extremely 
large  in  a  very  short  time,  their  rapid  growth  being  due  to  small 
pieces  of  nodule  tissue  or  to  clumps  of  bacteria  carried  over  into  the 
agar.  In  five  or  six  days  numerous  colonies  begin  to  make  their 
appearance,  most  of  them  as  submerged  colonies,  which  later  grow 
to  the  surface. 

"The  dilution-plate  (second  plate)  colonies  are  always  extremely 
slow  in  growth.  Generally  colonies  are  large  enough  for  transfer 
in  six  to  fourteen  days,  the  plates  should  not  be  discarded  for  two  or 
even  three  weeks. 

"  The  rate  of  growth  of  colonies  also  varies  with  the  organisms  of 
different  nodules.  Among  the  fast  growers  are  the  organisms  from 
pea  (Pisum),  vetch  (Vicia),  lentil  (Lens),  sweet  pea  (Lathy  rus), 
bean  (Phaseolus),  lupine  (Lupinus),  wild  bean  (Strophostyles), 
clover  (Trifolium),  sweet  clover  (Melilotus),  alfalfa  (Medicago), 
and  fenugreek  (Trigonella).  The  organisms  appreciably  slower  in 
growth  are  those  from  the  cowpea  (Vigna),  Japan  clover  (Lespedeza), 
tick  trefoil  (Desmodium),  acacia  (Acacia),  partridge  pea  (Cassia), 
false  indigo  (Baptisia),  dyer's  greenweed  (Genista),  peanut  (Arachis), 
soybean  (Glycine),  and  hog  peanut  (Amphicarpa) ." 

Morphology  of  the  Bacteria.— They  are  bacilli  and  when  full-grown 
vary  in  length  from  1  to  4  or  5/*.  It  is  not  uncommon  to  find  them 
from  0.5  to  0.6/i  wide  and  from  2  to  3ju  long  and  some  have  been 
found  to  measure  only  0. 18ju  wide  and  0.9/*  long.  The  bacilli  prevail 
in  the  youri|  nodule,  whereas  the  branched  forms  or  bacteroids 
predominate  in  the  older  structure.  In  the  cowpea  nodules  Burrill 
frequently  found  large  club-shaped  bacteroids,  though  the  branched 
forms  were  not  so  numerous.  The  bacteroids  are  best  demon- 
strated when  the  young  nodule  is  just  beginning  to  show  a  reddish 
interior.  At  this  stage  the  characteristic  x  and  y  forms  occur  in 
great  number  and  show  considerable  vacuolation  and  unevenness  in 
staining,  especially  when  stained  with  carbol-fuchsin. 

"In  the  old,  decomposing  nodule  the  bacteroids  are  extremely 
vacuolated  and  ghost-like,  showing  small,  oval,  deep-staining  bodies 
within.  The  inference  is  that  these  bodies  are  motile  swarmers 
which  later  free  themselves  from  the  ghost-like  capsules,  rather  than 
bud  off,  as  has  been  described  by  some  writers.  Frequently  the 
swollen  rods  have  a  beaded  appearance  with  unstained  bands  or 
areas.  A  few  motile  rods  may  sometimes  be  seen  in  hanging  drops 


BACTEROIDS  303 

in  this  stage,  and  sometimes  a  bacteroid  is  seen  to  oscillate  as  though 
swung  about  by  some  propelling  force  in  one  end.  Division  of  the 
bacteroids  into  bacilli,  as  represented  by  Dawson,  may  also  occur. 

"  When  first  plated  out,  the  young  colonies  consist  of  small  rods 
which  show  considerable  variation  in  length.  No  bacteriods  are 
present,  though  the  rods  are  sometimes  slightly  club-shaped  and 
sometimes  show  vacuolation.  However,  they  never  attain  the  size 
of  bacteroids.  With  frequent  transfers  the  rods  become  quite 
uniform  in  size  and  stain  deeply  and  evenly,  especially  with  anilin- 
gentian  violet. 

"  In  very  old  cultures  (three  months  on  ash  agar,  without  transfer) 
the  small,  oval  swarmers  and  the  normal  rods  predominate,  though 
a  few  club-shaped  and  a  few  branched  bacteroids  are  found.  The 
bacteroids  produced  upon  artificial  media  are  never  so  large  nor  so 
numerous  as  those  seen  in  mounts  direct  from  a  young  nodule. 

"Staining.— The  organisms  do  not  stain  well  with  ordinary 
aniline  stains.  Carbol-fuchsin  and  aniline-gentian-violet  (used 
steaming)  are  the  most  satisfactory  stains.  Though  carbol- 
fuchsin  was  preferred,  anilin-gentian-violet  stains  were  always 
used  as  checks,  because  the  former  stain  accents  the  vacuolated 
appearance,  particularly  in  bacteroids.  Carbol-fuchsin  is  especially 
useful  in  staining  bacteroids,  direct  from  the  nodule  and  also  old 
agar  cultures.  Kiskalt's  amyl-Gram  stain,  described  by  Harrison 
and  Barlow,  is  useful  since  the  amyl  alcohol  clears  up  the  field, 
leaving  the  bacteria  stained,  though  not  so  intensely.  This  stain, 
however,  should  not  be  considered  a  means  of  identifying  Ps. 
radicicola. 


FIG.  41. — Bacteroids,  showing  shape  and  occurrence  of  vacuoles.     (After    Whiting). 

"Bacteroids.— While  Ps.  radicicola  produces.no  spores,  it  produces 
bacteroids  which  are  very  evidently  more  resistant  than  the  normal 
rods.  Unfavorable  conditions,  such  as  unsuitable  media,  infrequent 
transfer,  or  addition  of  caffein  to  the  medium,  cause  their  appear- 
ance. This  is  in  accord  with  what  takes  place  in  the  nodule.  In  the 
growing  nodule,  when  development  is  most  rapid,  the  bacteroids  are 
at  their  maximum;  they  enable  the  organisms  to  multiply  rapidly 
in  spite  of  the  resistance  offered  by  the  plant  cells.  Transferred  to 
favorable  media  from  this  stage  the  normal  uniform  bacilli  are 
produced.  The  bacteroid  then  must  be  regarded  as  a  normal  and 


304  SYMBIOTIC  NITROGEN  FIXATION 

a  very  necessary  stage  in  the  life  of  the  organism.     Its  significance 
in  the  actual  fixation  of  nitrogen,  however,  is  pure  speculation." 

The  organisms  are  actively  motile  and  when  viewed  under  the 
microscope  may  be  seen  darting  about  with  amazing  rapidity,  now 
tumbling  end  over  end,  now  spinning  violently  on  the  short  axis 
and  then  sweeping  across  the  field  in  a  darting,  jerking  course. 
They  contain  from  6  to  20  flagella.  The  number  and  distribution 
of  the  flagella  are  variously  given  by  the  different  investigators  due 
probably  to  either  variation  in  organisms  or  to  the  difficulty  with 
which  flagella  is  demonstrated  owing  to  the  gum  or  slime  produced 
by  the  organism. 

Mode  of  Entrance  into  Host.— The  method  of  inoculation  and  the 
growth  of  the  nodule  is  described  as  follows  by  Whiting : 

"  As  the  tip  of  the  root  hair  of  the  legume  pushes  itself  out  into  the 
soil,  it  chances  to  come  into  intimate  contact  with  the  organism, 
B.  radicicola.  Some  scientists  have  exploited  the  view  that  the 
organism  is  attracted  to  the  plant  by  chemotaxis,  believing  that  the 
plant  excretes  a  substance,  probably  a  carbohydrate,  which  diffuses 
into  the  soil  solution  and  attracts  the  motile  organism.  While  it 
has  been  rather  definitely  shown  that  this  organism  progresses  in 
the  soil  at  a  rapid  rate,  nevertheless  the  number  of  root  hairs  infected 
is  too  small  to  lend  support  to  a  chemotactic  theory.  However  the 
case  may  be,  the  organisms  cluster  at  the  tip  of  the  hair  and  by 
means  of  an  enzyme  (or  otherwise)  rapidly  dissolve  the  cellulose  of 
the  cell  wall,  thus  enabling  the  organism  to  enter  the  root  hair.  As 
a  result,  there  is  a  decided  bending  of  the  tip,  causing  it  to  resemble 
a  shepherd's  crook.  This  was  early  observed  as  a  sign  of  complete 
infection.  It  is  claimed  that  other  root  hairs  which  form  after  infec- 
tion are  immune  to  the  attack  of  other  leguminous  bacteria. 

"The  organisms,  by  rapid  division  and  growth,  advance  through 
the  center  of  the  infected  root  hair.  Prazmowski  found  organisms 
in  the  cell  sap  and  even  in  the  epidermis  only  two  days  after  inocula- 
tion. In  this  advance  an  infection  strand  (Infektion-schlauche)  is 
formed,  which  consists  of  gelatinous  material,  and  in  the  earlier 
stages  of  development  this  strand  may  be  traced  from  the  root  hair 
into  the  inner  tissue  of  the  root  and  from  cell  to  cell  throughout  the 
nodule.  This  infecting  strand  is  not  supposed  to  constitute  a 
portion  of  the  living  tissue,  nor  is  it  a  well-defined  tube;  but,  as  Fred 
has  recently  shown,  it  consists  of  a  large  number  of  zooglea  occurring 
adjacent  to  one  another,  in  which  separate  bacteria  can  be  distin- 
guished. The  infecting  strand  branches  profusely  and  it  was  this 
habit  of  growth  which  caused  the  early  investigators  to  consider  it 
the  mycelium  of  a  fungous  growth. 

"Growth  of  the  Nodule.— The  presence  of  B.  radicicola  in  the 
tissues  of  the  root  causes  a  rapid  cell  division  in  the  pericycle. 
These  cells  become  larger  and  contain  more  protoplasm  than  the 


RELATIONSHIP  TO  HOST  305 

surrounding  cells,  and  as  growth  takes  place  the  cortical  parenchyma 
and  epidermis  are  forced  outward,  thus  forming  a  nodule.  The 
growth  of  the  nodule  is  apical.  The  various  tissues  common  to  the 
plant  are  present.  In  the  central  portion  of  the  nodule  is  the  so- 
called  bacteroidal  tissue,  which  is  ochre,  flesh,  or  gray  in  color, 
according  to  the  age  of  the  nodule,  and  in  this  portion  the  infecting 
strand  (Infektion-schlauche)  is  distinguished  in  the  young  nodule. 
It  ramifies  throughout  the  cells,  causing  those  which  it  enters  to 
lose  their  power  of  cell  division  but  not  of  growth.  Later,  or  in 
older  nodules,  the  infecting  strand  is  not  visible,  and  the  bacteroidal 
tissue  loses  its  firmness.  At  the  period  when  seed  formation  is  at 
its  height,  most  of  the  nodules  are  soft,  and  the  internal  tissues  slough 
off,  leaving  the  more  resistant  epidermal  tissue  a  mere  shell,  which 
later  decays.  The  endurance  of  the  nodule  depends  upon  several 
factors,— chiefly,  however,  upon  the  kind  of  legume  plant  on  which 
it  is  produced  and  the  need  of  nitrogen  by  that  plant. 

"Pierce  considers  the  nodules  as  originating  endogenously  from 
the  same  layer  of  cells  as  the  lateral  roots,  and  as  being  morpho- 
logically similar  to  them;  however,  as  the  lateral  roots  rupture  the 
epidermis  the  above  statement  is  not  entirely  in  accord  with  what 
actually  takes  place. 

"  The  nodules  are  largest  and  most  numerous  where  "  aeration  is 
best  in  the  soil.  In  saturated  soils  they  occur  at  the  surface  and 
are  often  found  colored  green,  very  similar  to  sunburned  potatoes. 
Nodules  form  in  solutions,  and  exceptionally  well  in  certain  nutrient 
solutions.  Several  interesting  instances  have  been  brought  to  the 
attention  of  the  Experiment  Station,  in  which  the  observers  believed 
that  the  nodules  had  grown  above  the  ground.  These  peculiarities 
were  undoubtedly  caused  by  unobserved  physical  conditions  occur- 
ring at  the  time  of  infection  or  afterward." 

Relationship  to  Host.— Even  today  the  relationship  between  Ps. 
radicicola  and  its  host  is  a  mooted  question.  Some  authors  claim  that 
they  are  true  parasites  and  that  the  relationship  between  the 
tubercle  organisms  and  their  host  plants  is  that  of  .two  contending 
parties  and  the  bacteria  draw  on  the  nitrogen  of  the  air  in  their 
endeavor  to  make  up  the  deficiency  of  nitrogenous  substances  which 
have  been  taken  from  them  by  the  plant.  Moreover,  inoculation 
experiments  have  demonstrated  that  Ps.  radicicola  causes  a  certain 
resistance  similar  to  that  produced  by  an  organism  in  combating  a 
true  parasite. 

Hiltner  has  given  the  six  following  conditions  as  instances  in  which 
immunity  demonstrates  itself: 

1.  The  organisms  cannot  get  into  the  plant. 

2.  The  organisms  gain  admission  into  the  plant,  but  do  not  pro- 
duce nodules  because  the  plant,  by  its  greater  resistance,  absorbs 
the  bacteria. 

20 


306  SYMBIOTIC  NITROGEN  FIXATION 

3.  The  organisms  enter  the  plant  and  produce  nodules,  but  no 
fixation  of  nitrogen  occurs. 

4.  The  organisms  enter,  produce  nodules,  and  nitrogen  is  fixed 
and  assimilated  by  the  plant. 

5.  The  organisms  are  so  efficient  in  comparison  with  the  plant 
that  the  latter  is  injured. 

6.  The  organisms  are  parasitic  and  the  plant  is  actually  killed. 
Certain  products  which  are  produced  by  the  invading  organism  in 

connection  with  the  host  have  been  taken  as  evidence  of  the  parasitic 
nature  of  the  bacteria,  whereas  others  consider  the  nodule  which 
forms  on  the  legume  root  a  result  of  irritation  due  to  a  parasite. 
Grieg-Smith,  however,  considers  the  formation  of  root  tubercles  not 
as  a  result  of  irritating  parasitic  action  but  rather  as  the  consequence 
of  the  production  of  nutrients  at  that  place  resulting  in  better 
nourishment  and  growth  of  the  cells  than  in  other  parts  of  the  roots. 

Fuhrmann  considers  that  the  fixation  of  atmospheric  nitrogen 
by  the  root-tubercle  organisms  begins  when  the  bacteroids  have 
reached  a  stage  when  they  are  colored  brown-red  by  addition  of 
tincture  of  iodine.  This  occurs  only  when  the  organisms  are  feeding 
almost  exclusively  upon  carbohydrates  and  the  available  nitrogen 
compounds  have  been  almost  completely  exhausted.  Many 
workers  prefer  to  call  the  relationship  up  until  this  stage  a  true 
parasitic  and  later  a  true  mutual  symbiosis. 

By  careful  staining  Fred  was  able  to  demonstrate  the  entering  of 
the  bacteria  through  the  root  hairs,  immediately  after  which  a 
tubercle  started  to  form.  A  series  of  sections  showed  that  mitosis 
goes  on  in  the  nodules  much  the  same  as  it  does  in  diseased  tissue  of 
animals.  The  mitotic  figures  are  larger,  very  irregular,  and  not  well 
marked  and  have  an  uneven  number  of  chromosomes.  In  the 
normal  roots  the  mitotic  figures  are  about  one-sixth  as  large,  very 
clear,  and  the  chromosomes  in  numerous  pairs.  This  he  considers 
bears  out  the  theory  that  the  legume  bacteria  are  symbiotic  parasites 
of  the  plant. 

If  we  accept  Whiting's  definition  of  mutual  symbiosis  "as  the 
contiguous  association  of  two  or  more  morphologically  distinct 
organisms  not  of  the  same  kind,  resulting  in  an  acquisition  of 
assimilated  food  substances  which  implies  that  the  organisms  con- 
cerned have  the  power  of  independent  existence,  but  that  both  are 
benefited  by  the  close  association,"  we  must  conclude  that  all  the 
evidence  bears  out  the  idea  that  the  relationship  existing  between 
Ps.  radwicola  and  legumes  is  one  of  mutual  symbiosis. 

Mechanism  of  Fixation  (Metabolism).— For  a  long  time  it  was 
believed  that  the  nitrogen  fixed  by  legume  bacteria  and  assimilated 
by  the  plant  was  obtained  through  the  leaves.  The  organisms  on 
the  roots  were  considered  to  in  some  way  stimulate  the  plant  so  that 
it  possessed  the  power  to  assimilate  nitrogen.  Stoklasa  considered 


MECHANISM  OF  FIXATION  307 

that  amids  were  first  formed  and  that  these  migrated  to  the  nodules, 
reacted  with  glucose  and  produced  protein  which  served  as  the 
nutrient  medium  for  the  bacteria.  In  this  connection  he  advanced 
the  idea  that  the  bacteria  produced  an  enzyme  which  enabled  the 
plant  to  fix  the  nitrogen.  This  theory,  however,  was  shown  to  be 
untenable  by  Whiting  who  grew  soybeans  and  cowpeas  under  careful 
control  conditions.  One  lot  received  a  definite  proportion  of  oxygen, 
and  carbon  dioxid,  a  second  oxygen  and  carbon  dioxid,  while  a  third 
received  ordinary  air.  He  found  that  these  plants  utilize  atmos- 
pheric nitrogen  through  their  roots  and  not  through  their  leaves. 

Nobbe  and  Hiltner  (1893)  considered  the  root  tubercles  to  be  the 
parts  of  the  leguminous  plants  where  the  free  nitrogen  is  assimilated 
and  that  the  direct  agents  of  the  assimilation  are  the  bacteroids  and 
not  the  bacteria  themselves.  As  to  the  metabolism  of  the  nitrogen 
by  these  bacteroids  the  ideas  at  present  are  very  indefinite.  Loew 
and  Aso  (1908)  suggested  that  ammonium  nitrite  was  the  first 
compound  produced,  the  nitrous  acid  being  readily  reduced-  to 
ammonia. 

Gautier  and  Drouin  considered  that  the  nitrogen  is  oxidized  to 
nitrous  and  nitric  acids,  whereas  Winogradsky  has  advanced  the  idea 
that  the  free  nitrogen  in  the  plasma  of  the  organism  may  unite  with 
nascent  hydrogen  and  form  ammonia  which  by  oxidation  would 
become  assimilable. 

Gerlach  and  Vogel  concluded  that  there  is  a  direct  union  of  free 
nitrogen  with  some  organic  compound  inside  the  bacterial  cell. 
Heinze  thinks  it  probable  that  nitrogen  is  at  once  brought  into 
combination  with  a  carbohydrate  (glycogen)  and  suggests  that  a 
salt  of  carbonic  acid  may  be  formed  first,  or  that  carbonic  acid  may 
be  produced  from  cyanamid.  All  of  these  theories,  however,  are 
purely  speculative  as  there  is  little  experimental  evidence  on  the 
subject. 

It  is  in  keeping  with  our  knowledge  of  bacteria  to  assume  that 
the  changes  are  catalyzed  by  enzymes  produced  by  the  bacteria, 
and  Hiltner  reported  the  findings  of  a  substance  which  is  produced 
by  the  legume  bacteria  which  can  dissolve  the  cell  wall  and  root 
hairs.  Yet  Beijerinck  claims  that  no  enzyme  has  been  found  which 
attacks  starch,  cellulose,  or  saccharose.  No  true  proteolytic  enzyme 
has  been  reported,  but  Benjamin  has  reported  the  presence  of  urease 
in  the  nodules  of  various  legumes.  This  enzyme  is,  however,  found 
quite  generally  in  plants  and  may  have  come  from  the  host  and  not 
the  bacteria.  Fred,  although  unable  to  detect  a  proteolytic  enzyme, 
has  obtained  evidence  of  the  presence  of  oxidases  in  the  slime  of 
various  legume  bacteria. 

There  are  two  main  suppositions  regarding  the  assimilation  of  the 
nitrogen  by  the  plant  as  follows :  (1)  That  the  bacteroids  are  bodily 
absorbed  by  the  plant  fluids;  and  (2)  that  the  bacteroids,  by  some 


308  SYMBIOTIC  NITROGEN  FIXATION 

sort  of  change,  produce  the  substance  containing  the  assimilable 
nitrogen  which  the  plant  used. 

There  appears  to  be  considerable  evidence  in  favor  of  this  second 
theory.  Stefan  thinks  that  the  transfer  of  the  assimilable  nitrogen 
from  the  organism  to  the  host  plants  follows  the  ordinary  physical 
laws  of  osmosis,  and  Golding  has  conducted  some  very  interesting 
experiments  on  the  removal  of  the  products  of  growth  in  the  assimila- 
tion by  nitrogen  by  legume  bacteria.  He  reasoned  that  the  plant 
played  an  important  role  in  the  removal  of  the  products  produced  by 
bacteria  in  the  nodules  aside  from  the  mere  furnishing  of  suitable 
food.  He  used  a  porous  Chamberland  filter  candle  placed  in  a 
culture  vessel  to  serve  to  imitate  natural  conditions.  The  parts  of 
the  plants  used  in  some  of  his  experiments  were  sterilized  in  order  to 
avoid  the  possibility  of  plant  enzyme  action.  As  a  result  of  his 
method  of  experimentation  he  obtained  a  much  greater  fixation  of 
nitrogen  than  other  experimenters  had  obtained.  He  concluded  that 
the  plant  plays  a  part  in  the  removal  of  soluble  products  of  growth, 
thus  permitting  a  more  rapid  reaction  than  where  the  products 
accumulate. 

The  results  of  Golding' s  most  extensive  experiment  are  sum- 
marized as  follows: 

Nitrogen  in 
,  Grams. 

500.0  gms.  of  stems  and  leaves.      . .  2.865 

26.2gnis.  of  roots  and  nodules  (quite  fresh) 0.094 

3000.0  c.c.  ammonia-free  distilled  water 0.000 

Total  nitrogen  to  start  with .2.959 

2870.0  c.c.  filtrates  and  drainings .  0.731 

566.2  gms.  wet  residue 2.570 

Total  nitrogen  after  experiment 3.301 

Total  gain  of  nitrogen  during  experiment        .    '.      .      .      .  0.342 

Attempts  have  been  made  to  obtain  an  insight  into  the  transforma- 
tion going  on  in  the  nodules  by  various  analyses.  These  have  been 
summarized  by  Whiting  as  follows : 

"  Chemical— The  chemical  composition  of  legumes  from  the 
standpoint  of  their  nitrogenous  constituents  has  been  investigated 
to  some  extent,  but  the  studies  closely  related  to  this  point  are 
relatively  few.  The  following  data  are  very  general  in  character 
and  relate  to  studies  concerning  the  total  nitrogen  content  of  the 
different  parts  of  legumes  at  different  periods  of  growth.  Studies 
upon  some  of  the  various  nitrogenous  compounds  are  also  included. 

"In  1895  Stoklasa,  working  with  lupines  (Lupinus  luteus  and  L. 
angustifolius) ,  found  that  the  nodules  were  richest  in  the  element 
nitrogen  at  the  time  of  blooming,  while  the  roots  appeared  to  be 
richest  in  that  element  at  the  fruiting  period.  His  results  are  given 
in  Table  I.  The  figures  for  the  nodules  indicate  the  nitrogen  is 
either  taken  up  by  the  plant  for  seed  production  or  diffused  into  the 
soil. 


CHEMICAL  309 

TABLE  I.— TOTAL  NITROGEN*  IN  LUPINUS  LUTETJS:  RESULTS  OBTAINED 
BY   STOKLASA.       (PERCENTAGE    ON  DRY   BASIS). 

Period.  Roots.  Nodules. 

Blooming 1.64  5.22 

.Fruiting 1.84  2.61 

Maturity 1.42  1.73 

"Stoklasa  also  determined  protein,  amids,  and  asparagin  in 
lupine  nodules.  The  protein  was  obtained  by  the  Stutzer  method, 
the  amids  by  the  Kjeldahl  method,  and  the  asparagin  by  calcula- 
tion from  the  ammonia  obtained  by  distillation  with  magnesium  oxid. 
Table  II  shows  his  results. 

TABLE    II.— NITROGEN    COMPOUNDS    IN    LUPINE    NODULES:    RESULTS 
OBTAINED   BY  STOKLASA.      (PERCENTAGE   ON  DRY  BASIS). 

Period.  Protein.  Amids.  Asparagin. 

Blossoming 3.99  0.35  0.34 

Maturity 1.54  0.15  Trace 

The  presence  of  asparagin  in  the  nodule  is  important,  as  it  is 
thought  to  be  intimately  related  with  the  formation  of  protein. 

"In  1901  Wassilieff  studied  the  nitrogen  compounds  in  white 
lupine  (Lupinus  alba)  seeds  and  seedlings.  He  found  that  the  seeds 
contained  7.68  per  cent,  of  total  nitrogen;  and  that  of  this,  6.89  per 
cent,  was  in  the  form  of  protein  and  0.53  per  cent,  was  precipitated  by 
phosphotungstic  acid,  leaving  a  difference  of  0.26  per  cent.,  aspara- 
gin. The  occurrence  of  asparagin  in  large  amounts  in  the  seed- 
lings is  shown  by  the  data  given  in  Table  III. 

TABLE  III.— NITROGEN  COMPOUNDS  IN  FOURTEEN-DAY-OLD  GREEN 
SEEDLINGS  OF  WHITE  LUPINES:  RESULTS  OBTAINED  BY 
WASSILIEFF  (EXPRESSED  IN  PERCENTAGE  ON  DRY  BASIS). 

Parts. 

Leaves  

Cotyledons 

Stems 

Roots 

Wassilieff  also  demonstrated  the  presence  of  leucin  and  tyrosin 
in  the  cotyledons  of  one-week-old  seedlings  of  white  lupines.  These 
and  other  amino-acids  would  be  expected  to  be  present  when  the 
protein  of  the  seed  is  breaking  down  for  the  nutrition  of  the  seedling. 

"Knisely  analyzed  the  leaves,  pods,  stems,  roots,  and  nodules 
of  lupine  plants  for  total  nitrogen  at  three  distinct  periods  of  develop- 
ment. His  results  show  better  than  the  others  presented  where  the 
nitrogen  accumulates  as  the  plant  matures. 

1  Phosphotungstic  acid. 


P.T.A.I 
nitrogen. 

Asparagin. 

Protein. 

Total 
nitrogen. 

0.53 

1.45 

4.11 

6.57 

0.63 

3.83 

2.44 

7.83 

0.42 

4.57 

1.56 

6.77 

0.46 

2.20 

1.87 

5.40 

310  SYMBIOTIC  NITROGEN  FIXATION 

TABLE    IV.— TOTAL   NITROGEN    IN    LUPINES:    RESULTS    OBTAINED    BY 

KNISELY.     (EXPRESSED  IN  PERCENTAGE  ON  DRY  BASIS). 

Period.  Leaves.  Pods.  Stems.  Roots.  Nodules. 

Full  bloom              .  .  4.02  3.07  1.15  0.92  5.17 

Pods  well  formed  .  .  3.70  3.38  0.88  0.83  3.29 

Pods  very  large      .  .  3.41  3.68  0.90  0.66  3.70 

"Schulze  and  Barbieri  examined  lupine  and  soybean  seeds  and 
seedlings  for  nitrogen  and  obtained  the  results  shown  in  Table  V. 

TABLE  V.— NITROGEN  IN  LUPINE  AND  SOYBEAN  SEEDS  AND  SEEDLINGS: 

RESULTS   OBTAINED   BY  SCHULZE   AND   BARBIERI    (EXPRESSED 

IN  PERCENTAGE   ON  DRY  BASIS). 

Total  P.T.A.  Filtrates 

Material.  nitrogen.  Protein.  nitrogen.          from  P.T.A. 

Lupine  seeds  ....  8.63  8.17  0.24  0.22 

Soybeans 6.73  6.32  0.13  0.28 

Lupine  dark  seedlings, 

eleven  to  twelve  days  old  10.64  3.40  1.60  5.64 

Lupine  dark  seedlings 

twelve  days  old  .  .  10.51  2.33  2.17  6.01 

Soybean  seedlings  fifteen 

days  old        ....        7.42  3.86  0.56  3.00 

"They  also  found  a  large  amount  of  asparagin  in  both  the  lupine 
and  the  soybean  seedlings. 

"Schulze  has  made  a  careful  study  of  the  compounds  in  plants, 
and  has  formulated  the  hypothesis  that  the  same  decomposition 
products  arise  from  protein  in  the  plant  as  outside  it,  but  that  in  the 
plant  the  compounds  are  further  altered,  thereby  affecting  in  varying 
degree  the  individual  products  of  the  hydrolytic  decomposition. 
A  comparison  of  the  analyses  of  pea  seedlings  one  week  old  and* those 
three  weeks  old  show  the  following  differences: 

Leucin.         Tyrosin.      Arginin.  Asparagin. 

1  week     ....     Abundant     Little       Present  Absent. 

3  weeks  ....      Much  less     Absent    Almost  absent        Very   abundant. 

Arginin  and  amido-acids  were  shown  to  be  present  in  the  lupine 
cotyledons,  but  asparagin  was  absent,  although  the  latter  substance 
was  found  in  the  stem  of  the  seedling.  It  has  been  suggested  that 
the  occurrence  of  asparagin  is  associated  with  the  disappearance 
of  amido-acids  and  not  of  protein.  Phenylalanin,  tyrosine,  and 
tryptophane  have  been  reported  in  the  white  lupine  (Lupinus  alba), 
tyrosin  and  tryptophane  in  vetch  (Vicia  sativa),  and  tryptophane 
in  the  garden  pea  (Pisum  sativum). 

"Smith  and  Robinson  found  4.19  per  cent,  of  nitrogen  in  soybean 
nodules  and  3.90  per  cent,  in  cowpea  nodules.  They  observed  that 
inoculation  increased  the  protein  content  of  soybean  plants  without 
increasing  the  yield  of  beans.  This  has  been  noted  by  other 
experimenters. 


CHEMICAL  311 

"Hopkins  has  reported  the  analyses  of  cowpea  plants  for  total 
nitrogen  with  and  without  inoculation.  The  nodules,  root,  and  tops 
were  analyzed  separately,  as  will  be  seen  by  reference  to  Table  VI. 

TABLE  VI.— NITROGEN  FIXATION  BY  COWPEAS:  RESULTS  OBTAINED 
BY  HOPKINS.     (EXPRESSED  IN  MGS.) 

Nitrogen 
Treatment.  Tops.  Roots.'         Nodules.  fixed. 

Ten  plants  with  bacteria    .      .      .  146  9                  11                  125 

Ten  plants  without  bacteria  38  3 

Ten  plants  with  bacteria    ...  171  10                 18                 140 

Ten  plants  without  bacteria  55  4 

Ten  plants  with  bacteria    ...  143  8                  17                  124 

Ten  plants  without  bacteria  40  4 

The  inoculated  plants  contained  a  much  greater  percentage  of 
nitrogen  than  the  uninoculated,  the  average  content  of  the  inoculated 
being  4.24  per  cent,  in  the  tops,  1.48  per  cent,  in  the  roots,  and  5.92 
per  cent,  in  the  nodules,  while  the  average  content  of  the  uninoculated 
was  2.48  per  cent,  in  the  tops  and  0.88  per  cent,  in  the  roots. 

"The  ash  and  the  ash  constituents  of  the  nodules  and  the  roots 
of  lupines  have  been  determined  by  Stoklasa,  as  presented  in 
Table  VII.  The  total  ash  of  the  nodules  was  found  to  be  6.32  per 
cent.,  while  that  of  the  roots  was  found  to  be  4.55  per  cent. 

TABLE    VII.— ASH    CONSTITUENTS   IN    LUPINE    NODULES   AND    ROOTS: 
RESULTS  OBTAINED    BY  STOKLASA.       (EXPRESSED    IN   PERCENTAGE.) 

Constituents.  Nodules.  Roots. 

Si 1.59  1.90 

S 4.90  6.38 

P 6.51  4.28 

K 17.31  12.05 

Na 16.94  19.94 

Mg     .      . 7.41  7.05 

Ca 7.64  12.04 

Fe 0.83  0.75 

"The  analyses  of  red-clover  nodules  show  a  potassium  content 
of  2.63  per  cent,  in  the  dry  matter.  The  nodules,  therefore,  are 
relatively  rich  in  mineral  elements  as  well  as  nitrogen  compounds; 
and  Stoklasa's  results  (See  Table  VII)  show  that  the  chief  differences 
between  the  roots  and  the  nodules  in  the  composition  of  the  ash 
constituents  are  in  phosphorus,  potassium,  calcium,  and  sodium. 
The  nodules  are  richer  in  the  first  two  elements  and  the  roots  in  the 
latter  two. 

"  In  brief,  the  chemical  data  which  have  been  considered,  although 
small  in  amount,  show  the  relative  richness  in  nitrogen  of  the 
nodule  as  compared  with  other  parts  of  the  plant.  They  point  to 
the  accumulation  of  nitrogen  in  the  seeds,  at  the  expense  of  the  other 
parts,  as  the  plant  matures.  That  the  nitrogen  exists  in  the  form 


312  SYMBIOTIC  NITROGEN  FIXATION 

of  protein,  asparagin,  and  other  soluble  forms,  is  also  clear.  The 
presence  of  various  aliphatic  and  carbocyclic  amino-acids  has  been 
mentioned." 

Sources  of  Energy.— Under  natural  conditions  the  legume  bacteria 
undoubtedly  obtain  the  energy  required  for  the  endothermic  reaction 
which  they  catalyze  from  the  plant  carbohydrates.  It  has  long  been 
known  that  decoctions  of  the  legumes  makes  the  best  media  on  which 
to  grow  these  organisms.  Temple  found  that  the  presence  of 
ground  alfalfa  caused  a  rapidtmultiplication  of  the  organisms  either 
in  solution  or  in  soil.  Grieg-Smith  found  dextrose,  levulose,  sac- 
charose, maltose,  and  mannite  to  furnish  a  good  source  of  carbon  for 
the  organisms,  but  lactose  was  a  very  poor  nutrient.  Temple  found 
saccharose  and  dextrose  superior  to  lactose,  whereas  he  found  levulose 
wholly  unsuited  to  their  needs. 

No  one  so  far  has  attempted  to  measure  their  energy  requirements 
when  growing  under  their  natural  symbiotic  condition.  Fred  has 
studied  their  growth  apart  from  the  host  plant  and  found  that  when 
considered  per  unit  of  carbohydrate  consumed  the  legume  bacteria 
fix  as  much  or  more  nitrogen  than  Azotobacter. 

"Aeration.— The  legume  bacteria  are  all  aerobic  and  the  nodules 
on  the  roots  of  the  plants  are  usually  near  the  surface.  Although 
nodules  will  form  on  plants  grown  in  water  cultures,  yet  they  are 
not  as  large  and  active  as  when  grown  in  a  well  aerated  soil.  The 
addition  of  oil  to  a  soil  or  water  culture  in  which  legumes  are  growing 
prevents  the  formation  of  the  nodules.  Moreover,  as  shown  by 
Whiting,  the  legumes  get  their  nitrogen  through  the  root  and  not 
the  leaves.  The  result  of  cultivation  of  legumes  is,  therefore, 
threefold:  (1)  The  loosening  up  of  the  soil,  thus  making  available 
to  the  nodule  bacteria  atmospheric  nitrogen  and  oxygen;  (2)  the 
working  of  the  soil  increases  other  bacterial  activity  which  in  turn 
renders  soluble  potassium,  phosphorus,  and  other  essential  elements 
in  the  soil;  (3)  the  loose  aerated  surface  tends  to  conserve  the 
moisture  of  the  lower  layers  which  can  be  drawn  on  by  the  plant, 
thus  making  more  nearly  optimum  moisture  conditions. 

Moisture.— The  root  systems  of  plants  vary  greatly  with  the 
moisture  content  of  the  soil.  Gain  found  that  legumes  grown  in 
moist  soil  spread  widely,  were  full  of  water,  became  covered  with 
root  hairs,  and  presented  a  large  surface  of  young  tissues.  In  the 
dry  soil  the  roots  were  less  spreading  and  the  epidermis  was  greatly 
thickened. 

In  moist  soil  the  tubercles  of  the  peas  were  scattered  all  over  the 
roots,  were  five  or  six  times  as  abundant  as  in  the  dry  soil,  and  were 
about  four  times  as  large  and  ovoid  in  shape;  while  in  the  dry  soil 
no  tubercles  were  produced  on  the  superficial  roots.  At  a  depth  of 
about  20  centimeters  some  tubercles  were  found  of  a  hemispherical 
shape  and  much  smaller  than  those  grown  in  moist  soil. 


INFLUENCE  OF  FERTILIZERS  313 

On  beans  about  twenty  times  as  many  tubercles  were  found  in 
the  moist  soil  and  microscopic  examinations  showed  important 
differences  in  the  number  of  bacteria  present  and  the  structure  of  the 
tubercles.  Similar  results  were  obtained  with  lupines  and  other 
plants.  This  is  what  is  to  be  expected,  for  when  the  root  system  is 
not  actively  functioning  the  nodules  are  slowly  destroyed  by  the 
nodule-forming  bacteria  within  and  the  saprophytic  organisms 
without.  The  nitrogen  fixed  by  the  plant  is  proportional  -to  the 
number  and  size  of  the  nodules.  Hence,  the  gains  made  in  combined 
nitrogen  are  dependent  upon  the  water  applied  to  the  legume. 
This  optimum  will  vary  with  different  soils.  Kalantarov  found  in 
a  loam  soil  that  nodule  bacteria  require  for  their  growth  a  minimum 
moisture  content  of  about  30  per  cent.,  whereas  Prucha  found  in  a 
sandy  soil  the  optimum  moisture  content  was  from  20  to  40  per  cent. 
Wilson  found  that  an  increase  in  moisture  content  from  35  to  45 
per  cent,  more  than  doubled  the  production  of  nodules,  while  with 
an  increase  from  45  to  55  per  cent,  it  was  nearly  doubled.  There- 
fore, water  is  necessary  for  the  normal  functioning  of  the  plant  and 
bacteria,  and  it  tends  to  leach  out  the  soluble  nitrogen  and  thus 
stimulates  to  greater  action  the  legume  bacteria,  for  it  is  known  that 
the  legume  feeds  first  on  the  combined  nitrogen  of  the  soil  and  turns 
to  the  atmosphere  only  when  this  is  greatly  reduced. 

Excessive  quantities  of  water  may  exclude  the  nitrogen  from  the 
roots  and  also  favor  anaerobic  action,  both  of  -which  would  be 
detrimental  to  the  legume  bacteria. 

Temperature.— It  is  a  well-known  fact  that  the  temperature  of  a 
soil  varies  with  its  moisture  content  and  a  water-logged  soil  is  slow 
to  heat  up.  Gain  found  the  temperature  of  moist  soil  at  midday 
to  be  7  degrees  cooler  than  dry  soil.  This  difference  in  temperature 
persists  throughout  the  day  and  night  and  is  noted  in  a  diminished 
degree  even  to  a  depth  of  four  feet.  This  difference  may  be  suffi- 
cient in  some  soils  to  greatly  decrease  or  increase  the  metabolism  of 
the  organism  which  Zipfel  finds  is  at  its  optimum  at  a  temperature 
between  18  and  20°  C. 

Influence  of  Fertilizers.— The  legume  bacteria  require  the  same 
elements  for  their  growth  as  do  other  plants,  and  the  application 
of  fertilizers  to  a  soil  which  increases  the  available  potassium  and 
phosphorus  is  attended  by  an  increased  bacterial  activity.  How- 
ever, it  has  long  been  known  that  nitrates  inhibit  nodule  formation. 
Wilson  found  that  nodule  development  was  prevented  by  the 
presence  of  nitrates,  sulphates,  and  ammonium  salts,  although  the 
organisms  retain  their  vitality  in  the  presence  of  these  salts.  It  is 
thought  by  some  that  the  addition  of  soluble  nitrates  to  the  soil 
decreases  by  a  kind  of  compensatory  action  the  formation  of  root 
tubercles  by  legumes.  Legumes  growing  on  soil  rich  in  nitrates 
may  actually  be  immune  to  the  nodule  bacteria  and  prevent  their 


314 


SYMBIOTIC  NITROGEN  FIXATION 


entrance  into  the  roots.     Small   quantities   of   nitrates   tend   to 
stimulate. 

Legumes  Associated  with  Non-legumes.— For  centuries  it  has  been 
the  practice  in  China,  Japan,  Western  Asia,  Northern  Africa,  as  well 
as  ancient  Rome,  to  grow  legumes  and  non-legumes  in  combination 
and  there  is  no  doubt  that  time  and  again  practical  farmers  have 
noted  the  more  vigorous  growth  and  darker  green  of  non-legumes 
when  so  grown,  long  before  the  investigations  of  Hellriegel  and  his 
associates  established  the  fact  that  leguines  are  capable  of  utilizing 
atmospheric  nitrogen.  His  discovery  gave  the  key  to  the  mystery, 


FIG.  42. — Double  pots  used  in  showing  influence  of  Legume  on  non-legume. 
(After  Lipman). 


—the  non-legume  was  getting  combined  nitrogen  from  the  legume. 
This  was  strikingly  demonstrated  by  a  very  ingenious  experiment 
conducted  by  Lipman.  He  grew  non-leguminous  plants  in  soil  in 
a  porous  pot  surrounded  by  earth  in  a  larger  glazed  earthenware  pot 
in  which  leguminous  plants  were  growing  and  found  that  under 
favorable  conditions  non-legumes  associated  with  legumes  may 
secure  large  amounts  of  nitrogen  from  the  latter.  This  power  of 
supplying  nitrogen  to  non-legumes  varies  <ty£B?  different  legumes. 
At  times  it  may  appear  as  an  increased  yield,  whereas  at  others  it 
may  appear  as  an  increased  proportion  of  nitrogen  in  the  dry  matter 
of  the  non-legume  or  both.  The  following  table  gives  the  per- 


SOIL  GAINS  IN  NITROGEN  315 

centages  of  increase  in  the  protein  content  and  length  of  leaf  in  the 
grasses  grown  with  clover  over  grasses  grown  alone  (Evans). 

Protein,  Length  of 

Kind  of  grass.  n   X  6.25.  leaf. 

Timothy  grown  in  lawn  plat 18.89  21.27 

Timothy  grown  in  field 7.68  19.41 

This,  however,  varies  with  the  soil  and  there  may  be  conditions 
in  which  not  only  the  protein  content  of  the  non-legume  is  slightly 
reduced  by  the  association  with  the  legume,  but  that  the  percentage 
of  nitrogen  in  the  legume  may  decrease  as  the  proportion  of  non- 
legume  in  the  mixture  increases  as  noted  by  Westgate.  Even  in 
these  cases  the  total  nitrogen  of  the  combined  crops  is  usually 
increased,  provided  the  legumes  are  properly  inoculated. 

Soil  Gains  in  Nitrogen.— The  gains  made  by  soil  in  nitrogen  are 
dependent  upon  a  number  of  factors. 

(1)  It  is  self-evident  that  the  soil  must  be  in  good  physical  condi- 
tion for  maximum  nitrogen  gains.  (2)  The  soil  must  contain  the 
elements  essential  to  plant  growth,  with  the  exception  of  nitrogen. 
The  law  of  minimum  holds  rigidly  in  this  case  and  the  gains  in  nitro- 
gen are  limited  by  the  limiting  element  of  plant  production  other 
than  nitrogen.  (3)  Soils  which  contain  sufficient  available  nitrogen 
for  the  production  of  good  crops  gain  little,  if  any,  nitrogen  from  the 
growth  of  legumes,  for  the  plants  together  with  the  bacteria  feed 
first  upon  the  combined  nitrogen  of  the  soil  and  only  upon  atmos- 
pheric nitrogen  when  the  soil  nitrogen  is  exhausted.  Soils  may 
contain  an  abundance  of  combined  organic  nitrogen  which  for  some 
reason  is  not  rendered  available  and  still  gain  from  the  turning  under 
of  properly  inoculated  legumes.  (4)  The  legume  must  be  properly 
inoculated;  otherwise  it  obtains  its  nitrogen  as  do  other  plants. 
(5)  The  soil  must  be  a  suitable  home  for  the  legume  and  bacteria, 
that  is,  it  must  have  a  correct  reaction,  moisture,  temperature,  and 
aeration  for  maximum  nitrogen  fixation.  Hence,  we  can  expect 
to  find  a  wide  divergence  in  the  results  reported  by  investigators. 

Frank  in  1891  found  that  soil  which  had  been  green-manured  with 
legumes  showed  an  appreciable  gain  of  nitrogen.  And  it  is  a  well- 
known  fact  that,  in  sand  culture  experiments  in  which  the  nitrogen 
of  the  soil  is  very  low  much  more  nitrogen  may  be  removed  in  the 
legume  crop  than  was  found  at  first  in  the  soil,  and  after  the  removal 
of  the  crop  the  soil  may  have  gained  in  nitrogen.  But  what  would 
happen  in  normally  productive  soil?  The  most  reliable  data  now 
existing  are  contributed  by  the  Illinois  Experiment  Station  and 
indicate  that  twro-thirds  of  the  nitrogen  in  legumes  grown  on  soils  of 
normal  productive  power  is  obtained  from  the  air.  These  figures  were 
obtained  from  the  analysis  of  inoculated  and  uninoculated  legumes 
from  like  areas  of  normal  soils  and  as  a  result  of  pot  experiments. 
Computed  by  these  data  a  four-ton  alfalfa  crop  adds  132  pounds,  a 


316  SYMBIOTIC  NITROGEN  FIXATION 

four-ton  crop  of  clover  adds  107  pounds,  a  four-ton  crop  of  cowpea 
hay  adds  115  pounds. 

These  are  the  quantities  of  nitrogen  which  reach  the  soil  under 
ideal  conditions,  but  some  may  be  lost  under  natural  conditions 
with  the  drainage,  and  possibly  by  other  means.  The  New  Jersey 
Experiment  Station  has  reported  a  gain  of  200  pounds  per  acre  where 
crimson  clover  has  been  grown,  whereas  the  Rhode  Island  Experi- 
ment Station,  as  a  result  of  pot  culture  experiments,  reports  a  gain  of 
400  pounds  per  acre  yearly.  This  experiment  extended  over  five 
years,  and  legumes  were  grown  both  in  the  summer  and  in  the 
winter.  The  tops  of  the  summer  legumes  (cowpeas  and  soybeans) 
were  removed  from  the  soil,  while  the  winter  legumes  (vetch)  were 
turned  back  into  the  soil. 

Shutt,  in  pot  and  plat  experiments  extending  over  two  years  in 
which  mammoth  red  clover  was  grown  on  soil  and  turned  under, 
showed  a  gain  of  179  pounds  of  nitrogen  per  acre  to  a  depth  of  9 
inches  in  the  pot  experiments  and  175  pounds  to  a  depth  of  4  inches 
in  the  plat  experiments.  A  light  sandy  loam  with  a  sandy  subsoil, 
when  planted  to  clover  continuously  and  reseeded  every  two  years, 
doubled  in  nitrogen  in  ten  years.  This  was  a  yearly  gain  of  nitrogen 
of  50  pounds  per  acre. 

Soil  Inoculation.— The  early  experiments  demonstrated  that 
legumes  assimilate  atmospheric  nitrogen  only  when  properly  inocu- 
lated. Since  that  time  much  has  been  written  on  soil  inoculation. 
However,  it  is'being  found  that  in  the  majority  of  cases  where  the 
physical  and  chemical  conditions  of  the  soil  are  optimum,  the 
ordinary  legume  bacteria  are  already  present.  This  is  especially 
true  where  that  particular  legume  has  been  grown  in  that  district 
for  some  time.  The  legume  organism  may  have  been  in  the  virgin 
soil,  having  come  from  the  native  legume,  or  carried  into  the  soil 
with  manure  or  dust.  When  a  new  legume  is  being  introduced  into 
a  district,  one  should  see  that  the  soil  is  properly  inoculated  for  the 
members  of  that  group.  Successful  seed  inoculation  can  be  per- 
formed with  fresh  properly  prepared  artificial  cultures,  but  in  many 
cases  this  has  not  proved  successful  and  in  the  majority  of  cases 
inoculation  with  soil  known  to  be  infected  is  to  be  preferred.  The 
method  suggested  by  the  Illinois  Experiment  Station  for  large  seeds, 
such  as  soybeans,  is  very  satisfactory.  The  seeds  are  thoroughly 
moistened  by  a  10  per  cent,  solution  of  glue  and  sufficient  dry 
pulverized  infected  soil  sifted  on  to  absorb  all  of  the  moisture.  The 
seed  is  shovelled  over  a  few  times.  Such  infected  seed  should  be 
planted  very  soon  or  else  spread  out  to  dry  to  prevent  mould  action. 
Neither  infected  seed  nor  soil  should  be  long  exposed  to  bright  sun- 
shine, as  this  is  very  destructive  to  the  bacteria. 

Where  the  old  plants  are  to  be  inoculated  a  few  hundred  pounds 
of  soil  may  be  obtained  from  an  old  infected  field  spread  on  the  new 
field  and  harrowed. 


ALTERNATIVE  METHOD  317 

Dr.  C.  B.  Lipman  gives  the  following  method  for  inoculating 
beans,  and  in  a  modified  form  it  can  be  used  for  other  legumes : 

"Method  Involving  the  Use  of  One  Commercial  Culture.— Prepare 
one-half  barrel  full  of  good  loam  soil  (150  pounds)  with  sufficient 
water  to  make  about  optimum  moisture  conditions.  This  soil  can 
be  kept  in  a  shallow  vat  about  a  foot  in  depth  or  in  some  other 
convenient  receptacle  where  it  can  be  well  aerated.  Purchase  one 
commercial  culture  from  any  of  the  commercial  firms  selling  legume 
bacterial  cultures,  choosing  a  culture  for  beans  of  the  variety  desired. 
The  amount  usually  sold  in  a  culture  for  one  acre  is  sufficient. 
Shake  this  up  with  a  few  quarts  of  boiled  water.  The  shaking  should 
be  continued  for  about  ten  minutes  to  get  all  the  bacteria  in  sus- 
pension. Pour  this  suspension  all  over  the  surface  of  the  soil  in  the 
vat  and  add  to  the  solution  about  one-quarter  of  a  pound  of  ordinary 
sugar  per  one  hundred  pounds  of  soil  used  in  the  vat.  This  should 
be  distributed  as  evenly  as  possible  through  the  soil  and  the  latter 
thoroughly  mixed  with  a  spade  or  hoe,  thus  distributing  both  the 
sugar  solution  and  the  culture.  After  that  the  inoculated  soil  is  to 
be  kept  in  a  warm  place  like  a  kitchen  or  a  warm  stable  and  the 
moisture  content  maintained  at  optimum  until  you  are  ready  to  use 
it  for  the  inoculation  of  beans  when  they  are  planted.  It  is  well  to 
allow  a  period  of  two  or  three  months  for  such  incubation. 

"At  the  end  of  the  incubation  period  or  when  getting  ready  to 
plant,  shake  up  some  of  the  inoculated  soil  with  clean  water  for 
a  few  minutes  as  before  to  get  a  good  suspension  of  the  bacteria. 
Pour  enough  of  this  suspension  over  the  bean  seed  in  large  tanks 
or  similar  receptacles  to  wet  the  seed  thoroughly,  but  not  enough 
to  allow  any  excess  of  water.  Then  spread  the  bean  seed  out  on  a 
canvas  in  a  thin  layer  in  the  shade.  As  soon  as  the  seed  is  air-dry  and 
will  not  stick,  place  in  a  planter  and  plant  immediately.  In  cases 
in  which  only  small  quantities  of  seed  are  to  be  planted,  the  suspen- 
sion need  not  be  made,  but  the  inoculated  soil  in  small  quantities 
can  be  mixed  with  the  seed  in  the  planter  and  allowed  to  drop  with 
the  beans  as  they  are  dropped  from  the  machine,  thus  introducing 
the  bacteria  into  the  soil  with  every  seed,  or  nearly  so. 

"Alternative  Method.— Where  it  is  not  desired  even  to  purchase 
one  commercial  culture,  inoculation  can  also  be  carried  out  entirely 
successfully  by  obtaining  soil  from  a  garden  in  which  beans  have 
grown  successfully  for  some  years  and  using  that  ,soil  for  making  up 
the  soil  suspension  or  for  mixing  with  the  seed  as  above  described. 
In  other  words,  this  garden  soil,  which  contains  the  necessary  bac- 
teria, will  serve  fully  as  well  as  the  inoculated  and  incubated  soil 
just  described  above.  This  is  of  course  the  simpler  method  to  those 
who  have  access  to  garden  soil  which  has  produced  beans  successfully. 
Soils  like  this  may  also  be  obtained  from  old  and  more  extensive 
bean  fields,  where  successful  bean-growing  has  been  carried  out. 


318  SYMBIOTIC  NITROGEN  FIXATION 

For  small  plots,  such  soil  can  be  directly  harrowed  into  the  soil  to 
be  inoculated  after  being  spread  (about  one  bushel  per  acre)  in  moist 
condition  on  a  cloudy  or  rainy  day." 

Commercial  Cultures.— Because  inoculation  by  means  of  soil  from 
old  fields  may  transmit  fungus  diseases,  weed  seed,  and  necessi- 
tates the  transference  of  large  quantities  of  soil  numerous  workers 
have  endeavored  to  inoculate  with  pure  cultures.  The  first 
attempt  was  made  by  Nobbe  and  Hiltner  who  placed  on  the  market 
a  preparation  called  "nitrogin."  Eight  kinds  were  prepared  suit- 
able for  the  different  legumes  and  were  sent  out  on  gelatin.  Some 
of  the  results  were  satisfactory,  but  on  the  whole  the  percentage  of 
failures  was  so  great  that  the  method  was  largely  discredited. 

Later  the  subject  was  investigated  by  Moore  who  considered  the 
failures  of  Nobbe  due  to  the  fact  that  he  had  grown  his  cultures  on 
gelatin.  This  contained  combined  nitrogen  in  abundance  and  the 
bacteria  lost  their  virulence  and  no  longer  possessed  the  power  of 
forcing  their  way  into  the  roots  of  leguminous  plants  and  producing 
nodules.  Moore  used  a  nitrogen-free  medium  for  the  growth  of 
his  cultures,  thus  increasing  their  nitrogen-fixing  power.  They 
were  distributed  on  cotton.  Later  liquid  cultures  were  used  and 
since  that  time  many  different  media,  including  even  soil  humus 
have  been  used  by  different  workers  with  varying  degrees  of 
success.  As  a  result,  at  the  present  time  some  commercial  cultures 
are  being  put  upon  the  market  which  are  probably  just  as  efficient 
as  are  the  natural-occurring  soil  organisms.  However,  some  work- 
ers claim  to  have  developed  organisms  possessing  a  high  physio- 
logical efficiency.  But  after  taking  all  the  facts  into  consideration, 
one  must  conclude  that  at  the  present  time  the  pure  cultures  have 
little  advantage  over  the  natural-occurring  organisms. 

REFERENCES. 

Lohnis:     Handbuch  der  Landwirtschaftlichen  Bakteriologie. 

Lafar:     Handbuch  der  Technischen  Mykologie. 

Voorhees  and  Lipman:  A  Review  of  Investigations  in  Soil  Bacteriology,  U..  S. 
Dept.  Agr.,  Off.  Exp.  Sta.  Bull.  194. 

Whiting:  A  Biochemic  Study  of  Nitrogen  in  Certain  Legumes,  Illinois  Exp. 
Sta.  Bull.  179. 

Burrill  and  Hansen:  Is  Symbiosis  Possible  between  Legume  Bacteria  and  Non- 
legume  Plants,  Illinois  Exp.  Sta.  Bull.  202. 


CHAPTER    XXV. 
CROP  ROTATION. 

FROM  time  immemorial  it  has  been  considered  a  self-evident  fact 
that  where  crop  rotation  is  practised  there  is  a  bigger  and  better 
yield.  The  farmers  of  ancient  Rome  understood  that  crops  fol- 
lowing beans,  peas  and  vetches  were  usually  better  than  those 
following  wheat  or  barley,  but  it  was  not  until  the  last  quarter 
of  the  nineteenth  century  that  it  was  learned  that  the  leguminous 
plants,  with  the  aid  of  associated  bacteria,  have  the  power  of  feeding 
on  the  free  nitrogen  of  the  air,  whereas  the  non-leguminous  plants 
have  not  this  power  and  require  a  supply  of  combined  nitrogen. 
Today  the  best  farmers  practise  some  system  of  crop  rotation. 
They  have  learned  from  experience  that  where  crop  rotation  is 
practised  the  crops  are  bigger  and  better  than  under  the  single 
crop  system.  This  is  usually  interpreted  as  indicating  that  crop 
rotation  has  increased  the  fertility  of  the  soil.  Many  , farmers 
plant  legumes  for  a  number  of  years  on  run-down  soil,  remove  the 
entire  crop  and  feel  confident  that  their  soil  is  becoming  richer  in 
plant-food.  Let  us  examine  some  of  the  results  obtained  in  care- 
fully planned  experiments  to  see  if  this  conclusion  is  warranted  by 
the  experimental  evidence. 

Essential  Elements.— Plants  are  composed  of  ten  elements,  each 
one  of  which  is  absolutely  essential  to  growth  and  seed  formation. 
Only  two— carbon  and  oxygen— are  secured  from  the  air  by  all 
plants;  only  one— hydrogen— from  the  water;  the  other  seven  are 
secured  by  all  plants  from  the  soil.  One  class  of  plants— the 
legumes— may,  under  appropriate  conditions,  obtain  their  nitrogen 
from  the  air.  Six  elements— phosphorus,  potassium,  magnesium, 
calcium,  iron  and  sulphur— are  obtained  from  the  soil  by  the  growing 
plant. 

Element  Added  by  Legumes.— The  great  majority  of  agricultural 
soils  contain  large  quantities  of  all  these  elements,  with  the  excep- 
tion of  nitrogen,  phosphorus  and  potassium.  These  are  used  by 
the  growing  plant  in  larger  quantities  than  are  any  of  the  other 
elements  which  are  obtained  directly  from  the  soil.  In  the  great 
majority  of  soils  nitrogen,  phosphorus,  or  potassium  is  the  limiting 
element  in  crop  production.  Therefore,,  our  problem  resolves 
itself  into  the  question :  Can  crop  rotation  maintain  these  elements 
in  the  soil  in  quantities  sufficient  for  maximum  yields?  Phosphorus 


320  CROP  ROTATION 

and  potassium  are  obtained  by  the  growing  plant  only  from  the 
soil;  it  is,  therefore,  self-evident  that  no  simple  system  of  crop 
rotation  can  maintain  the  phosphorus  and  potassium,  since  the 
quantity  within  the  soil  must  of  necessity  be  reduced  with  each 
crop  removed,  the  extent  depending  upon  the  specific  crop  grown. 
Hence,  nitrogen  is  the  only  element  which  we  can  hope  to  maintain 
by  crop  rotation.  But  this  is  the  element  which  is  found  in  the 
soil  in  smallest  quantity  and  removed  by  most  plants  in  larger 
quantities  than  the  phosphorus  or  potassium.  Moreover,  large 
quantities  of  this  element  are  at  times  lost  from  the  soil  by  leach- 
ing, while  the  loss  of  the  others  is  comparatively  small.  It  is  of 
the  greatest  importance,  therefore,  that  this  nitrogen  be  supplied 
to  the  soils  in  sufficient  quantities  for  crop  production  and  in  the 
cheapest  manner  possible.  The  total  quantity  of  these  three  ele- 
ments found  in  an  acre-foot  section  of  two  Utah  agricultural  soils, 
assuming  one  acre-foot  to  weight  3,600,000  pounds,  is  given  below: 

Greenville  Farm  (Utah),         Nephi  Farm  (Utah), 
pounds  per  acre.  pounds  per  acre. 

Nitrogen       . 4,904  3,744 

Phosphorus  .      .  2,700  8,388 

Potassium 60,560  87,840 

Both  soils  contain  an  abundance  of  potassium,  but  the  supply 
of  phosphorus  and  nitrogen  is  much  lower.  A  study  of  these 
results  shows  that  a  50-bushel  crop  of  wheat  each  year  for  forty- 
nine  years  would  remove  the  equivalent  of  the  total  quantity  of 
nitrogen  in  the  Greenville  soil  to  a  depth  of  one  foot,  while  a  similar 
crop  on  the  Nephi  farm  would  accomplish  this  in  just  thirty-seven 
years.  It  would,  however,  require  a  50-bushel  crop  one  hundred 
and  seventy  years  to  remove  the  phosphorus  from  the  Greenville 
soil  and  five  hundred  and  twenty-five  years  to  remove  it  from  the 
Nephi  soil.  Of  course  a  crop  would  never  remove  all  the  nitrogen 
or  phosphorus  from  a  soil,  but  in  actual  practice  the  elements  are 
slowly  removed,  the  crop  yields  beng  reduced  each  year  until 
a  certain  minimum  is  reached.  When  crops  can  no  longer  be 
produced  economically  then  the  owner  abandons  his  soil,  moves 
on  to  virgin  soils,  or  if  it  be  in  an  old  district  he  resorts  to  the 
expensive  commercial  fertilizer.  The  illustration  is,  however,  suffi- 
ciently accurate  to  make  it  clear  that  the  limiting  factor,  in  so  far 
as  soil  fertility  is  concerned  in  both  of  these  soils,  is  the  nitrogen. 
And  it  is  true  of  the  great  majority  of  all  soils  that  an  increased 
nitrogen  supply  means  an  increased  yield.  This  principle  is  one 
of  the  fundamentals  of  soil  fertility. 

Nitrogen.— Nitrogen  exists  in  the  atmosphere  in  inexhaustible 
quantities,  every  square  yard  of  land  having  seven  tons  of  nitrogen 
lying  over  it,  or  if  the  quantity  covering  one  acre  could  be  combined 
into  the  nitrate  it  would  be  worth  as  a  fertilizer  $125,000,000. 


ROTHAMSTED  ROTATION 


321 


Now  it  has  been  demonstrated  that  the  legumes— peas,  beans, 
alfalfa,  etc.— when  properly  infected  have  the  power  of  feeding  on 
this  limitless  supply  of  atmospheric  nitrogen,  while  the  non-legumes 
—barley,  wheat,  oats,  etc.— must  depend  upon  the  supply  within 
the  soil,  and  the  farmer  must  take  advantage  of  this  fact  to  supply 
nitrogen  for  his  crops,  as  the  commercial  fertilizer  cannot  be  eco- 
nomically used  for  the  production  of  most  crops,  as  is  seen  from  the 
fact  that  the  nitrogen  in  a  50-bushel  wheat  crop  would  cost  $14.40, 
or  20  tons  of  sugar-beets  $15.00,  or  1  ton  of  alfalfa  hay  $7.50,  if 
bought  as  a  commercial  fertilizer.  But  will  the  legume  draw  nitro- 
gen from  the  atmosphere  while  there  is  a  supply  in  the  soil,  or  will 
it  follow  the  line  of  least  resistance  and  turn  only  to  the  atmosphere 
when  nitrogen  is  lacking  in  the  soil  ?  If  it  does,  it  must  first  drain 
the  soil  of  its  valuable  nitrogen  and  thus  leave  it  no  richer  than  it 
was  before  the  legume  was  grown  upon  the  soil.  This  is  the  prob- 
lem which  this  chapter  is  to  answer. 

Rothamsted  Rotation.— Crop  rotation  'has  been  practised  for 
centuries,  but  the  oldest  system  on  which  we  have  accurate  infor- 
mation is  the  one  on  Agdell  Field  at  the  Rothamsted  Experiment 
Station.  This  system  was  inaugurated  in  1848  and  is  still  being 
carefully  followed.  It  consists  of  a  four-year  rotation,  as  follows: 

First  year:     Swede  turnips  (rutabagas). 

Second  year:     Barley. 

Third  year:     Clover  or  beans. 

Fourth  year:    Wheat. 

Still  another  system  has  been  running  parallel  and  similar  to 
this,  except  that  fallow  cultivation  is  practised  in  the  third  year 
instead  of  growing  a  legume.  The  average  yields  for  twenty-year 
periods  are  given  below.  These  systems  are  of  special  interest  to 
western  farmers,  for  when  we  substitute  sugar-beets  for  the  turnips 
and  alfalfa  or  peas  for  the  clover  or  beans,  we  have  nearly  an  ideal 
rotation  for  our  soils. 


Legumes.                                     Fallow. 

Crop. 

Yield  1st 

Yield  2d 

Yield  3d 

Yield  1st 

Yield  2d 

Yield  3d 

20  years, 

20  years, 

20  years, 

20  years, 

20  years, 

20  years, 

1848-68. 

1868-88. 

1888-08. 

1848-68. 

1868-88. 

1888-08. 

Turnips: 

Roots  (Ibs.)     .... 

5264.0 

1723.0 

967.0 

5785.0 

3067.0 

2502.0 

Leaves  (Ibs.)         .      .      . 

600.0 

447.0 

242.0 

629.0 

538.0 

458.0 

Barley: 

Grain  (bu.)     .      .      .      . 

38.0 

22.5 

13.7 

37.0 

22.8 

15.9 

Straw  (Ibs.)     .... 

2373.0 

1496.0 

1172.0 

2244.0 

1489.0 

1172.0 

Wheat: 

Grain  (bu.)      .... 

29.6 

21.1 

24.3 

34.5 

23.2 

23.5 

Straw  (Ibs.)     .... 

3169.0 

2082.0 

2445.0 

3761.0 

2420.0 

2412.0 

21 


322  CROP  ROTATION 

Even  where  the  legume  was  used  in  the  system  there  had  been 
a  decline  in  the  yield.  The  yield  of  the  turnips  during  the  first 
twenty  years  was  5264  pounds,  the  second  1723,  and  the  third 
only  967  pounds,  thus  showing  a  decrease  of  about  five-sixths  the 
original  in  sixty  years. 

The  results  with  the  barley  are  no  better,  for  there  is  a  drop 
from  the  fair  yield  of  38  bushels  per  acre  during  the  first  period  to 
only  13.7  during  the  third.  The  wheat  which  followed  the  legume 
in  the  rotation,  and  hence  occupied  the  most  favored  place  in  the 
system,  shows  a  decrease  of  5.3  bushels.  Not  even  a  good  yield 
has  been  maintained  for  the  clover,  for  from  1850  to  1874  the 
average  yield  was  4165  pounds,  while  from  1882  to  1906  the  yield 
was  only  1246  pounds.  In  reality  we  find  no  greater  decline  in 
the  yields  where  fallow  cultivation  is  practised.  But  both  sys- 
tems strongly  testify  to  the  fact  that  rotation  is  not  maintaining 
the  productive  powers  of  this  soil.  And  the  evidence  is  strong 
that  the  legume  gets  no  more  nitrogen  from  the  air  than  that 
which  is  removed  with  the  plant.  Otherwise,  we  should  expect 
better  results  in  the  legume  system  than  in  the  fallow  system. 

Nitrogen  Obtained  from  Atmosphere  by  Legumes.— That  the 
alfalfa,  when  grown  on  fertile  soil  and  the  crop  removed,  does  not 
increase  the  nitrogen  of  the  soil  is  seen  from  experiments  con- 
ducted by  Dr.  Hopkins  at  the  University  of  Illinois.  The  experi- 
ments were  made  possible  by  the  fact  that  many  of  the  Illinois 
soils  do  not  normally  contain  the  symbiotic  bacteria  which  make 
it  possible  for  the  alfalfa  to  obtain  nitrogen  from  the  air.  This 
being  the  case,  a  field  was  taken  which  had  not  grown  alfalfa  and 
which  did  not  contain  the  symbiotic  nitrogen-gathering  bacteria. 
This  was  planted  to  alfalfa,  only  one-half  of  it  being  inoculated 
with  the  legume  organism.  To  some  of  the  plots  were  added  lime 
and  phosphorus  to  make  sure  that  these  were  not  the  limiting  fac- 
tors. The  results  thus  obtained  are  given  below: 

Pounds  in  crop: 

Pounds,  nitrogen 


O.   UU.UUD,    AiiUlWfe^J-l 

Plot  No.  Treatment  applied.                       Dry  matter.      Nitrogen.         fixed  by  bacteria. 

lo          None 1180  21.81 

16          Bacteria 2300  62.04 

2a          Lime 1300  26.20                    ,,   S9 

26           Lime  bacteria 2570  68.02                 f 

3a  Lime  phosphorus  ....  1740  35.40 

36  Lime  phosphorus  bacteria      .  3290  89.05 

It  is  evident  from  these  results  that  the  alfalfa  had  obtained 
from  40  to  53  pounds  of  nitrogen  from  the  air,  depending  upon 
the  treatment.  There  was  slightly  more  than  one-third  as  much 
nitrogen  in  the  alfalfa  crop  from  the  uninoculated  as  in  the  inoculated, 
Therefore,  it  is  quite  evident  that  the  alfalfa  in  these  plats  had 
obtained  one-third  of  its  nitrogen  from  the  soil  and  two-thirds  frora 


LEGUMES  FEED  ON  NITRATES  323 

the  air.  Now,  nitrogen  is  required  by  the  root  for  its  growth  as 
well  as  for  the  growth  above  the  ground,  and  we  have  every  reason 
for  believing  that  the  root  also  would  obtain  it  in  the  same  pro- 
portion from  air  and  soil  as  did  the  hay  crop. 

Distribution  of  Nitrogen  in  Legumes.— If  we  examine  dry  matter 
and  total  nitrogen  occurring  in  the  roots  and  stalks  of  alfalfa,  we 
should  be  able  to  decide  whether  more  nitrogen  is  being  returned 
to  the  soil  in  the  roots  and  residues  than  is  removed  by  the  growing 
plants. 

The  results  for  this  comparison  have  been  obtained  from  Illinois 
and  Delaware  experiments  and  are  tabulated  below: 


T.  egume. 
Sweet  clover: 
Tops 

Dry  matter 
per  acre, 
pounds. 

.      9029 

Nitrogen 
per  acre, 
pounds. 

174  \ 

Per  cent,  of 
total  nitrogen, 
in  tops. 

Roots  and  residues    . 
Crimson  clover: 
Tops 

.      .      .      3748 
.      .      .      4512 

54  / 
103  X 

Roots  
Alfalfa: 
Tops 

.      .      .      2022 
2267 

41  / 
54.  8  \ 

Art 

Roots  

1980 

40.  4  / 

With  the  clover,  three-fourths  of  the  total  nitrogen  is  found 
in  the  plant  above  ground  and  only  one-fourth  in  the  roots,  while 
alfalfa  shows  a  greater  proportion  in  the  roots— 40  per  cent.  This 
represents  the  proportion  for  the  first-year  growth  for  alfalfa  and 
it  is  not  likely  that  in  the  older  plant  this  proportion  of  the  total 
nitrogen  would  be  maintained  in  the  roots.  Hence,  it  is  quite 
certain  that  if  only  two-thirds  of  the  total  nitrogen  of  the  plant 
is  obtained  from  the  air  the  quantity  returned  to  the  soil  with  the 
roots  and  plant  residues  does  not  exceed  that  removed  from  the 
soil  by  the  growing  plant,  which  would  give  no  increase  in  soil 
nitrogen  from  the  growing  of  a  legume  where  the  entire  crop  is 
removed,  and  this  even  where  the  roots  are  allowed  to  remain 
and  decay.  Yet  we  find  some  farmers  who  remove  the  roots  from 
the  soil  and  even  then  expect  an  increase  in  their  soil  fertility. 

Legumes  Feed  on  Nitrates.— It  is,  therefore,  rather  certain  that 
the  legume,  where  the  crop  is  harvested,  does  not  increase  the 
soil  nitrogen  of  the  fertile  soil  of  Illinois  and  other  soil  fairly  rich 
in  nitrogen.  But  what  will  happen  on  the  arid  and  semi-arid  soil 
where  nitrogen  in  many  cases  is  the  limiting  element  and  is  present 
in  much  smaller  quantities  than  it  is  in  the  soils  on  which  the 
experiments  considered  have  been  conducted.  Experiments  which 
have  been  conducted  at  the  Utah  Experiment  Station  during  the 
last  twelve  years  have  demonstrated  that  even  on  soils  poor  in 
nitrogen  the  legume  first  feeds  upon  the  combined  nitrogen  of  the 
soil.  It  is  known  that  plant  residues  and  other  complex  nitrogen 


324  CROP  ROTATION 

compounds  found  in  the  soil  are  transformed  by  bacteria  into 
ammonia,  and  this  in  turn  by  another  class  of  bacteria  into  nitric 
nitrogen,  and  it  is  mainly  on  this  nitrogen  that  the  growing  plant 
feeds.  The  quantity  of  this  found  in  the  soil  at  different  periods 
under  different  plants  has  been  measured  at  the  Utah  Experiment 
Station  and  the  average  results  for  twelve  years  are  given  in  tabular 
form  below,  stated  as  pounds  of  nitric  nitrogen  per  acre  to  a  depth 
of  six  feet. 

Season. 

Crop.  Average. 

Spring.       Midsummer.         Fall. 

Alfalfa 22.3  15.8  32.8  23.6 

Oats     .            35.7  14.1  20.6  23.5 

Corn    .      . 24.8  18.9  22.0  21.9 

Potatoes 81.1  60.8  54.2  65.3 

Fallow 81.5  53.6  62.6  65.9 

The  legume,  alfalfa,  removes  the  nitric  nitrogen  from  the  soil 
equally  as  fast  as  do  the  non-legumes.  Yet  this  soil  was  well- 
inoculated  with  the  symbiotic  bacteria  which  undoubtedly  assisted 
the  alfalfa  in  obtaining  free  nitrogen  from  the  air  when  needed,  but 
not  until  the  soluble  nitrogen  had  been  drained  from  the  soil  to 
its  full  extent,  as  shown  by  the  fact  that  alfalfa  soil  never  contains 
more  than  does  oat  and  corn  land,  and  is  very  poor  as  compared 
with  potato  and  fallow  soil. 

Nitrification  in  Soils. — It  may  be  argued  that  the  small  quantity 
of  nitric  nitrogen  in  the  alfalfa  soil  is  due  to  a  lack  of  its  formation, 
as  it  is  not  needed  by  the  legume,  and  hence  not  formed.  This 
conclusion,  however,  is  not  warranted  by  the  facts  in  the  case,  as 
may  be  seen  from  the  results  obtained  where  nitrification  was 
measured.  These  also  are  the  average  results  extending  over  a 
number  of  years  and  obtained  at  the  Utah  Experiment  Station. 

Milligrams,  nitric  nitrogen  produced 
in  100  gms.  of  soil  in  twenty-one  days. 

Crop.  • • >          Average. 

Spring.      Midsummer.         Fall. 

Alfalfa 3.15  7.48  3.08  4.56 

Oats 2.40  4.00  3.00  3.13 

Corn 2.18  3.50  1.48  2.38 

Potatoes    .      .            ....  3.00  15.55  5.60  8.04 

Fallow 1.30  5.50  2.48  3.09 

Here  the  quantity  of  soluble  nitrogen  produced  in  the  alfalfa 
soil  is.  greater  than  that  produced  in  either  the  oat,  corn,  or  fallow 
soil.  There  is  no  doubt  that  this  is  one  reason  why  an  increased 
yield  is  obtained  the  year  following  the  plowing  up  of  legumes  for  this 
increased  action  also  occurs  the  next  year  after  an  alfalfa  field  is 
planted  to  some  other  crop.  This  is  due  to  the  stimulation  of 
bacterial  organisms  of  the  soil  by  the  alfalfa  plant  so  that  they 
make  available  faster  the  nitrogen  of  the  soil,  but  this  only  depletes 
the  soil  of  its  nitrogen  more  readily  than  the  non  legume,  as  it  is 
the  nitrogen  already  combined  in  the  soil  on  which  the  nitrifying 


HOW  TO  MAINTAIN  SOIL  NITROGEN  325 

organisms  act.  Hence,  we  must  conclude  that  alfalfa  not  only 
feeds  closer  on  the  soluble  nitrates  of  the  soil,  but  it  also  makes  a 
greater  drain  upon  the  insoluble  nitrogen  of  the  soil  by  increasing 
its  nitrifying  powers  and  would  therefore  deplete  the  soil  if  the 
entire  crop  be  removed,  more  readily  than  would  other  crops— a 
conclusion  which  is  borne  out  by  the  direct  analysis  of  the  soil. 

The  analysis  of  a  great  number  of  Utah  soils  which  have  grown 
various  crops  for  a  number  of  years— some  of  them  having  been 
into  alfalfa  or  wheat  for  upward  of  thirty  years — revealed  the 
fact  that  almost  invariably  the  alfalfa  soil  contained  less  total 
nitrogen  than  did  the  wheat  soil.  The  average  for  a  great  number 
of  determinations  made  from  alfalfa  soils  was  7232  pounds  per 
acre  of  total  nitrogen,  while  the  average  for  a  great  number  of 
wheat  soils  was  7398  pounds.  These  are  average  results  from  a  great 
number  of  determinations  made  on  adjoining  alfalfa  and  wheat  soil 
and  they  clearly  indicate  that  in  ordinary  farm  practice  the  alfalfa 
is  making  just  as  heavy  a  drain  upon  the  soil  nitrogen  as  is  the 
wheat. 

Hence,  from  a  consideration  of  the  yields  obtained  in  crop  rota- 
tion, the  relative  quantities  of  nitrogen  obtained  from  the  atmos- 
phere and  the  soil  by  the  alfalfa,  the  feeding  and  stimulating  effect 
of  the  alfalfa  upon  nitrification,  and  finally  the  actual  quantity  of 
total  nitrogen  remaining  in  the  soil  after  wheat  and  legumes^  we 
must  conclude  that  the  legume  does  not  increase  the  nitrogen  of 
a  common  agricultural  soil— even  in  the  arid  region  where  the 
nitrogen  is  low— when  the  entire  crop  is  removed. 

This  conclusion  does  not,  however,  mean  that  crop  rotation 
should  not  be  practised,  for  there  are  many  reasons  why  crop  rota- 
tion commends  itself  to  the  careful  farmer,  but  it  must  not  be  used 
and  the  legume  removed  with  the  intention  of  maintaining  soil 
fertility.  This  may  appear  to  be  an  unfortunate  conclusion,  but 
it  is  just  the  reverse,  and  if  its  teachings  be  heeded  it  means  a 
fertile  soil  and  an  economic  gain  to  the  farmer  from  the  system  of 
farming  which  it  requires  him  to  adopt. 

How  to  Maintain  Soil  Nitrogen.— There  are  two  practicable 
methods  of  maintaining  the  nitrogen  content  of  the  soil.  (1) 
Planning  systems  of  crop  rotations  with  legumes,  the  legumes 
being  plowed  under  and  allowed  to  decay,  thus  furnishing  nitrogen 
to  the  succeeding  crop;  (2)  practising  a  combined  system  of  crop 
rotation  and  livestock  farming. 

Three  tons  of  alfalfa  contain  150  pounds  of  nitrogen,  all  of 
which  we  could  assume  came  from  the  atmosphere.  Assuming 
the  quantity  found  in  the  roots  as  coming  from  the  soil,  this  is  the 
equivalent  of  the  nitrogen  found  in  the  grain  and  straw  of  75 
bushels  of  wheat.  If  the  alfalfa  is  plowed  under  some  of  the 
nitrogen  would  be  lost  to  the  growing  plant  in  the  processes  of 
decay  and  leaching,  but  that  the  total  nitrogen  of  the  soil  may 


326  CROP  ROTATION 

actually  be  increased  by  the  turning  under  of  the  legume  is  certain 
from  field  experiments. 

The  Dominion  of  Canada  Experiment  Stations  grew  mammoth 
clover  for  two  successive  seasons  on  a  soil  very  low  in  nitrogen. 
The  two  cuttings  of  mammoth  clover  with  all  the  residues  were 
turned  under  each  year  with  the  result  that  the  soil  gained  as  an 
average  177  pounds  per  acre  of  total  nitrogen  which  is  the  quantity 
of  nitrogen  found  in  three  40-bushel  crops  of  wheat,  provided  the 
straw  was  returned  to  the  soil,  as  two  tons  of  this  contains  20 
pounds  of  nitrogen.  On  the  other  hand,  work  on  the  soil  of  the 
Utah  Nephi  Experiment  Farm,  with  a  rotation  of  wheat  and  peas 
where  the  peas  were  plowed  under,  showed  a  gain  in  total  nitrogen 
of  240  pounds  in  four  years.  That  is,  in  addition  to  furnishing  the 
small  quantity  of  nitrogen  required  by  the  wheat  crop,  the  peas 
had  added  to  the  soil  an  average  of  60  pounds  of  nitrogen  per  year. 

The  second  method  of  maintaining  the  nitrogen  and  organic 
matter  of  the  soil— the  combined  rotation  and  livestock  method— 
is  the  more  practical,  and  if  systematically  practised  will  not  only 
maintain  the  nitrogen  of  the  soil  but  will  prove  of  great  economic 
value  to  the  individual  following  it.  For  it  consists  of  a  rotation 
in  which  the  legume  plays  a  prominent  part.  The  legume  to  be 
fed  and  all  the  manure  returned  to  the  soil,  which  would  mean  the 
selling  from  the  farm  of  the  hay  crop  in  the  form  of  butter,  milk  or 
beef  which  carries  from  the  soil  only  a  fraction  of  the  nitrogen 
stored  up  by  the  legume.  Moreover,  it  brings  for  the  producer 
much  greater  returns  than  does  the  system  in  which  the  legume  is 
plowed  under. 

It  must,  however,  be  remembered  in  this  system  that  only  three- 
fourths  of  the  total  nitrogen  of  the  feed  is  recovered  in  the  dung 
and  urine.(  So  that  in  place  of  three  tons  of  alfalfa  adding  150 
pounds  of  nitrogen  to  the  soil  from  the  air,  it  would  add  only  120 
pounds,  and  this  is  where  all  of  the  liquid  and  solid  excrements  are 
collected  and  returned  to  the  soil.  But  where  the  alfalfa  is  to  be 
fed  and  the  manure  returned  to  the  soil,  the  legume  can  occupy 
a  much  longer  period  in  the  rotation  and  that  with  greater  economy 
than  where  the  legume  is  to  be  plowed  under  directly. 

Hence,  we  find  that  if  these  principles  which  have  been  estab- 
lished for  soils  even  low  in  nitrogen  be  systematically  applied  to 
the  soil,  it  will  result  in  greater  revenue  from  an  increased  live- 
stock industry  and  will  maintain  the  soil  rich  in  nitrogen  and 
organic  matter  in  place  of  depleting  it  of  its  stored-up  nitrogen, 
as  is  so  often  the  case  with  the  present  methods. 

REFERENCES. 

Hopkins:     Soil  Fertility  and  Permanent  Agriculture. 

Greaves,  Stewart  and  Hirst:  Influence  of  Crop,  Season  and  Water  on  the  Bac- 
terial Activities  of  the  Soil,  Jour.  Agr.  Research,  ix,  293-341. 


CHAPTER  XXVI. 
CELLULOSE-DECOMPOSING  ORGANISMS. 

THE  plant  residues  which  find  their  way  into  the  soil  contain, 
in  addition  to  protein,  non-protein  compounds.  These  are  decom- 
posed by  microorganisms,  thus  liberating  the  energy  and  returning 
the  carbon  to  the  atmosphere  so  that  it  is  again  available  to  plants. 
The  reactions  occurring  in  this  process  are  probably  the  reverse 
of  those  occurring  in  the  fixation  of  carbon  by  the  plant. 


Plant 


CO2     +     H2O 

I 
sugars 

I 
starches 

4 

cellulose 


absorbed     Microorganisms 


CO2     +     H2O 

t 

sugars 

T 

starch 

T 

cellulose 


energy 
liberated 


Cellulose.— The  term  cellulose  does  not  designate  a  single  indi- 
vidual compound  but  undoubtedly  a  whole  series  of  compounds. 
All  of  these  are  extremely  complex  and  pass  gradually  from  the 
tender  hemi-  or  pseudo-cellulose  of  the  young  plant,  which  is 
comparatively  soluble  in  acids  and  alkalies,  to  the  more  complex 
and  very  resistant  lignocelluloses.  All  are  forms  of  cellulose,  but 
their  properties  are  exceedingly  different.  The  first  may  serve  as 
food  even  to  man,  but  the  latter  is  highly  resistant  to  all  the  common 
solvents.  It  is,  however,  dissolved  by  a  few  special  solvents,  such 
as  ammoniacal  solutions  of  copper  oxid,  carbon  bisulphid  in  sodium 
hydroxid,  and  a  few  others.  Cellulose  is  nitrogen-free  and  is 
made  up  of  carbon,  hydrogen,  and  oxygen  having  the  empirical 
formula,  (CeHioOs)^  On  hydrolysis,  it  yields  various  sugars, 
depending  upon  its  source,  as  glucose,  mannose  or  xylose.  In 
the  process  of  hydrolysis,  there  results  certain  intermediate 
dextrin  bodies,  a  study  of  which  has  shown  cellulose  to  be  ex- 
tremely complex.  Besides  these  there  are  certain  gums,  pectins, 
lignins  and  similar  compounds,  which  are  nearly  related  to  cellu- 
lose and  which  have  not  been  differentiated  from  the  true  cellulose 
by  many  investigators.  The  results  are  that  the  power  of  decom- 
posing cellulose  has  been  attributed  to  certain  organisms  but  a 
careful  study  of  the  subject  has  revealed  later  that  the  organism 
decomposed  some  of  the  related  compounds  but  left  cellulose 
unaltered. 

Early  Observations.— That  carbon  passes  through  a  definite  cycle 
from  the  solid  organic  tissues  of  plants  to  the  gaseous  form  of  the 


328  CELLULOSE-DECOMPOSING  ORGANISMS 

atmosphere  has  been  known  for  a  long  time,  but  it  was  usually 
thought  of  as  passing  from  the  solid  complexes  to  the  gaseous 
compounds  through  its  direct  combination  with  oxygen  at  a  high 
temperature.  In  fact,  this  was  considered  as  being  the  only 
method  until  Pasteur  pointed  out  that  there  were  other  means. 
He  considered  it  as  being  brought  about  by  molds.  Later  Mit- 
scherlich  (1850)  observed  that  when  moist  potatoes  decay  the  cell 
wall  is  dissolved  and  the  starch  gradually  passes  out.  This  he 
thought  to  be  due  to  a  group  of  organisms,  but  nothing  was  done 
to  show  that  it  was  the  work  of  any  species  until  about  fifteen 
years  later  when  Trecul  isolated  an  organism  which  had  the  power 
of  decomposing  young  plant  tissues  and  which  was  stained  blue 
by  iodine.  To  this  organism  he  gave  the  name  amylobacter. 
The  organism  he  claimed  had  the  power  of  decomposing  cellulose 
with  the  formation  of  butyric  acid,  carbon  dioxid,  and  hydrogen. 
As  all  of  his  work,  however,  was  carried  on  with  plant  tissues,  it 
leaves  a  question  as  to  whether  the  amylobacter  had  actually 
decomposed  cellulose  or  only  some  of  the  nearly  related  compounds. 

The  decomposition  of  cellulose  in  manure  was  studied  by  Dehe- 
rain,  Gayon,  Herbert  and  Popoff.  The  last  investigator  was  the 
first  to  recognize  the  similarity  between  the  method  of  production 
of  methane  in  sewage  and  the  intestines  of  animals.  He  studied 
the  action  which  took  place  when  a  medium  containing  Swedish 
filter  paper  was  seeded  with  sewage,  and  obtained  a  large  volume 
of  gas,  an  analysis  of  which  showed  it  to  consist  of  carbon  dioxid, 
methane  and  hydrogen.  The  first  two  he  thought  to  be  due  to 
a  cellulose  ferment,  but  the  latter  to  a  butyric  acid  ferment.  At 
the  end  of  the  incubation  period,  there  was  a  gummy  mass  in  the 
fermentation  flasks. 

For  a  long  time  after  this  the  attention  of  the  investigators 
seemed  to  be  directed  mainly  to  a  quantitative  study  of  the  result- 
ing products  of  fermentation.  This  is  especially  true  with  the 
work  of  Tappeiner  and  Hoppe-Seyler.  The  former,  with  the  idea 
of  determining  the  bacterial  changes  which  take  place  normally  in 
the  intestinal  canal,  introduced  finely  divided  cotton  or  paper  into 
flasks  containing  a  1  per  cent,  neutral  solution  of  beef  extract. 
The  flasks  and  contents  were  sterilized  and  then  inoculated  with 
small  quantities  of  pancreatic  juice  and  incubated  at  35°  C.  They 
were  so  arranged  that  the  gases  could  be  collected  and  analyzed. 
The  resulting  product  consisted  of  acetic  acid,  isobutyric  acid, 
acetaldehyd,  methane  and  carbon  dioxid.  The  last  two  were 
in  the  ratio  of  1  to  7.2  at  the  beginning  of  the  process  and  1  to  3.4 
at  the  close.  In  another  set  of  experiments  he  used  an  alkaline 
medium  and  obtained  the  same  qualitative  but  different  quanti- 
tative results,  there  being  a  large  amount  of  hydrogen  evolved  in 
the  alkaline  medium. 


EARLY  OBSERVATIONS  329 

From  his  work,  he  concluded  that  cellulose  undergoes  a  fer- 
mentation in  the  first  stomach  of  ruminants  and  in  the  alimentary 
canal  of  all  herbivora.  In  later  work  he  tried  to  decide  whether 
this  fermentation  was  due  to  an  organized  or  to  an  unorganized 
ferment.  This  he  did  by  inoculating  suitable  flasks  with  the 
contents  of  the  alimentary  canal  of  oxen.  The  flasks  were  divided 
into  three  sets  and  treated  as  follows:  (1)  Heated,  (2)  treated 
with  antiseptics  (thymol  and  the  like)  and  (3)  untreated.  Fer- 
mentation occurred  only  in  the  last  set  from  which  he  concluded 
that  it  was  due  to  bacterial  action.  From  his  work  in  general 
he  decided  that  bacteria  have  the  power  of  decomposing  cellulose 
with  the  formation  of  carbon  dioxid  and  methane  and  that  this 
process  plays  a  large  part  in  the  digestive  processes  of  herbivorous 
animals. 

Hoppe-Seyler,  who  considered  the  fermentation  process  mainly 
from  the  changes  which  take  place  when  cellulose  is  decomposed 
in  soil  or  beneath  water,  commenced  his  experiments  by  collecting 
and  analyzing  the  gases  given  off  from  soils  and  swamps.  These 
he  found  to  consist  mainly  of  carbon  dioxid  and  methane.  Later 
he  carried  out  laboratory  determinations  by  placing  25.773  grams 
of  filter  paper  into  1000  c.c.  flasks  containing  700  c.c.  of  water  and 
inoculated  with  mud.  They  were  so  arranged  that  the  gaseous 
products  were  collected  over  mercury.  He  incubated  them  at 
room  temperature  for  four  years.  During  the  first  year  there  was 
considerable  gas  evolved,  but  the  evolution  gradually  became 
slower  until  at  the  end  of  four  years  the  evolution  of  gas  had  practi- 
cally ceased.  An  analysis  showed  that  15  grams  of  the  cellulose  had 
been  decomposed  with  the  formation  mainly  of  carbon  dioxid 
and  methane.  He  was  unable  to  find  any  of  the  true  sugars, 
although  he  thought  it  possible  that  there  were  some  of  the  dextrin 
compounds  in  the  solution.  When  air  was  excluded  he  found 
that  there  was  a  greater  production  of  methane  and  a  smaller  one 
of  carbon  dioxid.  From  his  work  he  considered  the  reaction  pro- 
ceeded in  two  stages:  First,  a  hydration  of  the  cellulose  with 
the  formation  of  a  hexose  according  to  the  equation,  C6Hi005+ 
H2O  ->  C6Hi2O6.  From  the  hexose,  carbon  dioxid  and  methane 
was  formed  (C6Hi2O6  -»  3C02  +  3CH4),  or  perhaps  acetic  acid 
was  an  intermediate  product  and  then  carbon  dioxid  and  methane 
were  formed  according  to  the  equation,  CH3COOH  ->  C02  +  CH4. 

In  1889  Schlosing  published  his  quantitative  results  of  the 
investigation  on  the  decay  of  manure.  He  collected  the  gases 
given  off  in  the  course  of  two  months  in  the  decay  of  manure  and 
analyzed  them.  He  concluded  that  the  change  was  similar  to 
alcoholic  fermentation. 

Three  years  later  the  work  of  Herbert  appeared.  He  inoculated 
5  per  cent,  solutions  of  potassium  carbonate  or  ammonium  car- 


330  CELLULOSE-DECOMPOSING  ORGANISMS 

i 

bonate  containing  finely  divided  straw  with  manure.  At  the  end 
of  three  months,  when  the  evolution  of  carbon  dioxid  and  methane 
had  nearly  ceased,  he  examined  the  residue  with  the  following 
results : 

At  beginning  At  end 

Substance  in  the  straw.  of  experiment.  of  process.  Loss. 

Cellulose 14.12  6.18  56.2 

Wood  gum 10.00  4.67  53.3 

Vasculose 14.01  11.75  16.1 

Deherain  studied  the  substances  given  off  in  the  decomposition 
of  manure  with  the  following  results: 

Top  layer  of  Middle  of  Bottom  of 

manure  heap,  manure  heap,  manure  heap , 

per  cent.  per  cent.  per  cent. 

Carbon  dioxid 21.6  31.0  37.1 

Oxygen 0.0  0.0  0.0 

Methane 0.0  33.3  58.0 

Nitrogen 78.4  35.5  4.9 

In  the  layers  in  which  there  was  considerable  oxygen,  as  may 
be  seen,  the  amount  of  combustible  gas  given  off  was  zero,  but  in 
the  middle  and  lower  layers  of  the  manure  heap  the  resulting 
methane  was  over  half  of  the  gaseous  product.  Similar  results 
were  obtained  by  Gayon,  who  studied  the  changes  resulting  with 
a  limited  and  free  access  of  air  and  found  that  methane  was  obtained 
in  much  larger  quantities  when  the  air  had  been  excluded.  From 
this,  he  concluded  that  methane  fermentation  is  due  to  an  anaerobic 
organism. 

Preceding  this  work  was  that  of  van  Senus,  who  found  that 
cotton  and  plant  tissues  were  decomposed  by  microorganisms  with 
the  formation  of  carbon  dioxid,  methane,  hydrogen,  butyric  acid, 
acetic  acid,  alcohol,  aldehyd  and  a  trace  of  the  higher  fatty  acids. 
He  thought  the  methane  was  formed  through  the  reduction  of 
acetic  acid  by  means  of  hydrogen.  He  considered  the  action  as 
being  brought  about  by  two  organisms— one  the  amylobacter  of 
Trectil,  and  another  very  small  bacillus  which  he  had  isolated  from 
the  alimentary  canal  of  a  rabbit.  He  considered  that  they  acted 
by  means  of  an  excreted  enzyme,  which  he  precipitated  by  means 
of  alcohol  and  found  an  aqueous  solution  of  the  same  had  the 
power  of  decomposing  cellulose. 

Work  of  Omelianski.— As  may  be  seen  from  the  preceding  brief 
summary  of  the  work,  practically  all  that  had  been  done  on  the 
subject  prior  to  1895  was  directed  at  a  study  of  the  chemistry  of 
the  process  and  little  had  been  done  in  trying  to  isolate  in 
pure  cultures  the  specific  organism  or  organisms  which  had  the 
property  of  decomposing  cellulose.  It  was  at  this  point  that  the 
work  was  taken  up  by  Omelianski,  who  studied  very  carefully  the 
chemical  and  bacteriological  phases  of  cellulose  fermentation. 


MORPHOLOGY  AND^  PHYSIOLOGY  331 

In  his  work,  the  following  medium  was  used: 

Potassium  phosphate l"gm. 

Magnesium  sulphate 1     ' 

Ammonium  sulphate 1 

Sodium  chlorid trace 

Distilled  water 1000  c.c. 

In  some  cases  he  replaced  the  ammonium  salt  with  0.5  per  cent, 
asparagin,  1  per  cent,  peptone,  or  0.5  per  cent,  beef  extract.  The 
solutions  were  placed  in  flasks  containing  filter  paper  and  then 
inoculated.  Inasmuch  as  the  incubation  period  of  cellulose  fer- 
mentation is  long  and  variable,  he  found  it  best  to  seed  with  con- 
siderable of  the  organism.  Ordinarily,  this  was  done  by  taking 
a  small  piece  of  the  decomposing  paper  from  an  old  culture. 

Soon  after  inoculation  there  was  observed  a  slight  turbidity 
in  the  flasks.  Then  the  paper  thickened  and  assumed  a  decayed 
appearance.  It  was  covered  with  little  specked  places  where  it 
had  been  decomposed  by  the  organisms.  This  latter,  appearance 
varied;  at  times  the  holes  were  large  and  few,  and  at  other  times 
they  were  small  and  very  numerous.  At  still  other  times  the 
paper  seemed  to  thicken  and  then  to  fall  to  pieces.  At  the  end  of 
the  process  there  remained  a  residue  entirely  different  from  the 
original  paper.  In  old  cultures  the  white  appearance  of  the  paper 
had  disappeared  and  it  had  taken  on  a  yellowish  brown  color, 
which  often  appeared  even  in  the  surrounding  solution.  The 
gases  given  off  had  the  odor  of  decayed  cheese.  At  the  height  of 
the  process  particles  of  filter  paper  were  carried  to  the  surface  of 
the  liquid  by  the  gas.  The  above  description  applies  to  the  process 
as  brought  about  by  both  the  hydrogen  and  methane  organ- 
isms which  Omelianski  succeeded  in  isolating  in  pure  cultures  by 
the  method  of  repeated  heating  (75°  C.  for  fifteen  minutes),  which 
is  based  on  a  difference  in  the  life  history  of  the  two  organisms. 
The  methane  fermentation  organism  develops  more  rapidly  than 
the  other  variety  and  gains  the  supremacy  in  the  early  stages  of 
the  process.  If  heat  be  applied  at  this  stage  the  more  slowly  germi- 
nating spores  of  the  hydrogen-fermenting  organism,  being  in  a 
resistant  stage,  survive^ 

Morphology  and  Physiology.— A  microscopic  examination  of  the 
hydrogen  ferment  reveals  the  following:  In  the  young  culture  the 
bacillus  is  about  0.5  IJL  in  width  and  from  4  to  8  /z  in  length.  In 
old  cultures  they  are  from  10  to  15  IJL  in  length.  They  never  occur 
linked  together  in  chains  but  appear  as  slightly  bent  rods.  "Err 
still  older  cultures  they  take  the  drumstick  form  which  gradually 
develops  into  a  round  spore  about  1.5  IJL  in  diameter.  They  are 
readily  stained  by  the  anilin  dyes.  In  old  cultures  they  give  the 
characteristic  colors  for  the  spore  and  surrounding  sheath  with 
carbol-fuchsin  and  methylene  blue.  They  are  not  stained  blue 


332  CELLULOSE-DECOMPOSING  ORGANISMS 

with  iodin,  and  consequently  are  different  from  the  amylobacter 
of  Trecul.  No  growth  occurs  usually  in  the  ordinary  cultural 
media,  though  Omelianski  has  observed  on  some  occasions  very 
minute  translucent  colonies  on  potatoes. 

This  investigator  carried  out  quantitative  determinations  of  the 
substances  yielded  by  the  organism.  It  was  done  in  flasks  con- 
taining 300  c.c.  of  a  mineral  salt  solution  containing  calcium  car- 
bonate and  Swedish  filter  paper.  The  flasks  were  inoculated 
with  the  organism  and  incubated  for  thirteen  months.  On  analysis 
the  following  results  were  obtained: 

Resulting  products. 

Cellulose  at  beginning  of  process     3 . 4743  Fatty  acids  2 . 2402 

Cellulose  at  end  of  period    .      .      0.1272  Carbon  dioxid  0.9722 

Decomposed 3.3471  Hydrogen  0.0138 

Chief  among  the  fatty  acids  yielded  were  acetic,  butyric,  and 
valeric  acid.  Besides  these  there  were  traces  of  the  higher  acids 
found. 

The  methane  fermentation,  according  to  Omelianski,  takes 
place  if  a  flask  containing  filter  paper,  lime  and  a  mineral  neutral 
solution  be  inoculated  with  mud  or  fresh  horse  manure  and  kept 
under  anaerobic  conditions  at  a  temperature  of  from  35°  to  37°  C. 
After  a  short  time  a  careful  examination  of  the  filter  paper  revealed 
numerous  bacilli.  By  successive  subculturing,  while  the  methane 
fermentation  was  at  its  height,  the  hydrogen  ferment  was  soon 
eliminated.  The  methane  organism  is  rod-shaped,  slightly  more 
bent  than  the  hydrogen  ferment.  It  never  develops  in  chains, 
but  in  old  cultures  assumes  the  typical  drumstick  form  with  a  spore 
in  the  end.  The  organism  is  0.4  /*  in  width  and  5  /*  in  length.  It 
is  not  stained  blue  by  iodine  and  hence  is  different  from  the  amylo- 
bacter of  Trecul.  From  this  it  may  be  seen  that  both  the  vegetative 
cell  and  spore  are  slightly  smaller  than  the  hydrogen  ferment. 
Though  morphologically  very  similar,  physiologically  they  are 
very  different,  since  one  yielded  hydrogen  and  the  other  methane. 
This  is  shown  by  the  quantitative  studies  of  Omelianski.  They 
were  conducted  in  500  c.c.  flasks  containing  2.0685  gms.  of  Swedish 
filter  paper,  4.9482  gms.  of  calcium  carbonate,  and  a  0.1  per  cent, 
solution  of  ammonium  sulphate.  They  were  inoculated  with  0.013 
gm.  of  filter  paper  from  an  old  culture.  Over  one  month  elapsed 
before  fermentation  became  perceptible  and  even  then  it  was  very 
slow  as  is  shown  by  the  fact  that  the  gaseous  material  evolved 
never  exceeded  1.1  c.c.  in  twenty-four  hours,  and  at  the  end  of 
four  and  a  half  months  had  dropped  to  0.01  c.c.  for  twenty-four 
hours.  The  resulting  gas  was  mainly  carbon  dioxid  and  methane, 
0.7146  gm.  of  the  carbon  dioxid  and  0.1372  gm.  of  the  methane. 
In  the  flask  there  remained  only  a  small  amount  of  cellulose  but 


FUNCTION  333 

a  large  amount  of  acetic  and  butyric  acids.  In  fact  over  one-half 
of  the  decomposed  cellulose  had  been  transformed  into  these  acids. 

Later  Work  on  Cellulose  Fermentation.— Later  work  which  has 
been  carried  out  by  vanxlterson  has  shown  that  there  are  certain 
of  the  non-sporeforming,  denitrifying  organisms  which  have  the 
power  of  decomposing  cellulose.  In  the  presence  of  nitrates,  the 
chief  products  are  nitrogen  and  carbon  dioxid.  According  to  this 
investigator,  the  decomposition  is  brought  about  by  Bacillus  ferru- 
gineus,  which  is  the  chief  cause  of  the  brown  color  of  humus  due 
to  a  pigment  formed  from  cellulose  by  this  organism. 

Recently  Kellermann  and  McBeth  have  questioned  the  work  of 
Omelianski.  While  they  admit  the  great  importance  of  these 
organisms  in  the  formation  of  humus  in  agricultural  soils,  they 
claim  that  the  organisms  described  by  Omelianski  were  not  pure 
cultures  and  furthermore  that  cellulose  is  decomposed  under 
aerobic  conditions.  These  investigators  have  isolated  thirty-six 
species  from  various  sources.  These  were  found  to  be  much  more 
active  than  the  ones  described  by  Omelianski.  They  were  all  rod- 
shaped  organisms  varying  in  length  from  0.8  to  3.5  //.  Involution 
forms  have  been  observed  for  only  three  species.  Five  species 
have  been  found  to  produce  spores.  Twenty-seven  species  are 
motile;  of  these,  seven  are  pseudomonas  and  twenty  are  bacilli. 
A  few  are  facultative  anaerobes.  The  optimum  temperature  lies 
between  28°  and  33°  C.,  but  they  grow  well  from  20°  to  37.5°  C. 
They  grow  readily  on  solid  media  such  as  beef  agar,  gelatin,  starch 
and  potato.  Nineteen  species  liquefy  gelatin.  They  rapidly 
decompose  cellulose  and  other  carbohydrates  with  the  production 
of  acids,  but  none  of  the  organisms  so  far  studied  produce  a  gas. 

Function.— It  may  be  well  to  call  attention  to  the  great  part 
taken  by  this  class  of  organisms  in  returning  carbon  to  the  atmos- 
phere. This  is  especially  the  case  with  the  material  which  passes 
off  in  the  sewage.  In  septic  tanks  there  are  millions  of  these 
organisms  busy  changing  the  most  resistant  organic  matter  into 
gaseous  products,  and  many  large  cities  today  depend  upon  this 
for  the  disposal  of  their  sewage.  Organisms  also  take  a  great  part 
in  the  purification  of  a  city's  water  supply.  They  also  take  part 
in  the  formation  of  soil  humus,  as  was  pointed  out  by  Omelianski 
when  he  gave  the  general  reaction,  2C6Hi005  — » 5CO2  4-  SCHi  -f  2C, 
and  he  says,  "It  is  possible  that  a  general  reaction  of  this  sort 
forms  the  basis  of  the  universal  processes  of  humification,  that  is, 
the  gradual  transformation  of  organic  substances  into  a  mixture 
of  brown  and  black  substances  with  a  high  content  of  carbon,  such 
as  is  characteristic  of  fossil  coals.  But  whatever  the  mechanism 
of  these  transformations,  the  active  participation  of  microorgan- 
isms in  the  latter  cannot  be  denied." 

The  cellulose  ferments  break  the  plant  residues  into  less  com- 


334  CELLULOSE-DECOMPOSING  ORGANISMS 

plex  organic  compounds  which  are  fermented  by  other  organisms 
with  the  generation  of  large  quantities  of  organic  acids.  These 
would  react  with  the  minerals  of  the  soil  rendering  them  available. 
This  is  very  likely  the  cause  of  the  good  results  obtained  from  raw 
rock  phosphate  and  stable  manure  on  phosphorus-poor  soil.  The 
fermentation  of  the  cellulose  yields  acids  which  render  soluble 
the  phosphorus.  This  formation  of  acids  may  at  times,  however, 
become  excessive,  giving  rise  to  the  sour  humus  of  moors  and 
heaths. 

It  is  well  known  that  the  fermentation  processes  in  the  soil 
resulting  in  the  decomposition  of  organic  matter  may  give  rise  to 
large  quantities  of  carbon  dioxid,  methane  and  hydrogen.  The 
hydrogen  and  methane  do  not  all  pass  into  the  atmosphere,  but 
according  to  the  researches  of  recent  investigators  furnish  energy 
to  numerous  soil  organisms,  the  importance  of  which  remains 
almost  wholly  for  future  workers  to  develop.  The  first  work  on 
this  subject  was  done  by  Immendorff,  who  in  1892  found  that 
hydrogen  and  oxygen  may  be  made  to  unite  under  the  influence  of 
soil.  He  found  that  the  oxidation  of  hydrogen  was  brought  about 
only  by  normal  soil  and  not  by  soil  previously  treated  with  chloro- 
form vapor.  This  observation  remained  unnoticed  until  recently 
when  two  papers  appeared— one  by  Kaserer  and  the  other  by 
Solmgen— which  throw  considerable  light  on  this  phase  of  carbon 
and  hydrogen  transformation.  They  used  an  inorganic  solution 
containing  dipotassium  phosphate,  ammonium  chlorid,  magnesium 
sulphate,  sodium  bicarbonate,  and  a  trace  of  ferric  chlorid.  This, 
they  inoculated  with  a  small  quantity  of  'soil  and  confined  in 
an  atmosphere  consisting  of  a  mixture  of  hydrogen,  oxygen  and 
carbon  dioxid.  Growth  took  place  and  the  hydrogen  disappeared. 
The  presence  of  a  small  quantity  of  carbon  dioxid  seemed  to  be 
necessary  for  the  development  of  the  organisms,  and  it  would 
appear  that  like  the  nitrifying  bacteria  they  can  produce  bacterial 
protein  in  inorganic  solutions,  deriving  their  carbon  from  carbon 
dioxid.  This  reaction,  according  to  Lipman,  is  of  great  significance 
in  agriculture,  for  a  great  loss  of  energy  is  prevented  by  the  bacterial 
oxidation  of  hydrogen  formed  in  the  deeper  layers  of  the  soil  by 
anaerobic  ferments.  It  also  partly  counteracts  the  rapid  minerali- 
zation of  organic  materials,  in  that  it  leads  to  the  formation  of  com- 
plex compounds  from  carbon  dioxid,  hydrogen  and  oxygen. 

Kaserer  and  Solmgen  also  obtained  organisms  capable  of  utilizing 
methane  as  the  sole  source  of  energy  in  their  life  process.  The 
latter  investigator  secured  pure  culture  of  an  organism  which  he 
named  Bacillus  methanicus.  When  grown  in  inorganic  solutions 
confined  in  an  atmosphere  of  one-third  methane  and  two-thirds 
air,  it  caused  the  disappearance  of  the  methane  with  the  production 
of  considerable  quan^es  of  organic  material. 


FUNCTION  335 

The  cellulose  ferments  also  perform  other  direct  functions  in 
the  soil,  as  for  instance,  the  liberating  of  plant  food  which  is  bound 
up  in  plant  residues.  Heinze  has  very  recently  ascribed  to  bacterial 
activities  much  of  the  benefits  obtained  from  summer  fallowing. 
In  quantitative  studies  he  found  them  to  be  more  numerous  in 
fallow  soil  than  in  cropped  soil,  and  he  thinks  it  to  be  due  to  their 
activities  in  rendering  the  cellulose  more  suitable  as  a  carbon 
supply  for  the  Azotobacter  that  causes  the  increase  of  soil  nitrogen 
in  fallow  land  noted  by  a  number  of  recent  workers.  One  of  the 
more  important  problems  of  today  in  soil  bacteriology  is  the  rela- 
tionship between  this  class  of  organisms  and  other  important  soil 
organisms,  especially  the  nitrifiers  and  the  nitrogen  fixers.  » 

REFERENCES. 

'  Lafar:     Handbuch  der  Technischen  Mykologie. 
McBeth:     Studies  on  the  Decomposition  of  Cellulose  in   Soils,  Soil   Science,  i, 

437-488. 


CHAPTER   XXVIII. 
BACTERIA  IN  AIR. 

BACTERIA  require  for  their  growth  moisture,  food,  a  suitable 
temperature  and  usually  the  absence  of  direct  sunlight.  The 
moisture  conditions  of  the  atmosphere  at  times  may  be  optimum 
for  the  growth  and  reproduction  of  bacteria,  but  none  of  the  other 
conditions  are.  Hence,  bacteria  do  not  multiply  and  grow  in  the 
atmosphere  as  they  do  in  water,  soil  and  milk.  These  substances 
may  and  do  have  a  natural  bacterial  flora,  but  we  cannot  consider 
the  air  as  having  a  definite  one,  for  the  number  and  kind  continually 
vary  with  many  factors  and  there  are  scarcely  two  places  having 
the  same  number  and  species  of  microorganisms. 

How  Bacteria  Enter  Air.— The  earth  is  surrounded  by  the  atmos- 
phere, which  when  "  looked  at  as  a  whole,  its  calms  are  exceptional, 
and  its  movements  are  the  rule.  We  may  find  the  gentle  breeze, 
the  cyclonic  wind  or  the  restless  tornado,  but  always  active.  These 
movements  do  not  confine  themselves  to  horizontal  paths,  but  the 
gases  rise  and  plunge,  pursue  broad  curves  and  narrow  spirals,  and 
would  present— to  an  eye  that  could  see  them  from  above— a 
tumult  like  the  sea  in  storm."  In  all  this  activity  it  is  picking 
up  bits  of  sand,  organic  matter  and  oftentimes  even  water.  These 
contain  microorganisms  which  are  carried  into  the  air  and  may 
subside  with  the  particle  to  which  they  adhere  or  become  free  and 
float  about  for  a  period. 

As  the  waters  of  the  ocean,  lakes,  rivers  and  smaller  streams  beat 
against  some  barrier  the  fine  spray  so  formed  carries  into  the  air 
bacteria,  as  do  also  the  hurrying  feet  and  rattling  wheels  in  a 
crowded  street.  Furthermore,  individuals  speaking  or  coughing 
force  from  the  mouth  numerous  bacteria  which  for  a  time  help  to 
make  up  the  microbial  inhabitants  of  the  atmosphere. 

Number  and  Kind.— The  number  and  kind  of  organisms  found 
in  the  air  are  governed  largely  by  the  locality.  They  are  most 
plentiful  in  densely  populated  areas  and  within  buildings  such  as 
churches,  theaters  and  other  places  where  a  large  number  of  people 
congregate.  Miquel  found  that  as  an  average  1  cubic  meter  of  air  from 
the  streets  of  Paris  contained  3480  bacteria,  laboratory  air,  7420, 
the  air  of  old  houses  36,000,  whereas  the  air  of  the  Paris  Hospital 
contained  79,000  bacteria  in  1  cubic  meter.  It  is  quite  evident  from 
these  figures  that  air  of  inhabited  districts  contains  many  bacteria. 
These  are  carried  on  the  dust  particles.  It  does  not,  however, 
always  follow  that  the  number  of  bacteria  in  the  air  is  an  exact 


FACTORS  GOVERNING  NUMBER  AND  KIND  337 

measure  of  the  number  of  dust  particles.  An  examination  of 
the  air  of  the  London  streets  showed  it  to  contain  between  300,000 
and  500,000  dust  particles  per  cubic  centimeter,  but  there  was 
only  one  microorganism  to  every  38,300,000  dust  particles.  The 
number  of  species  present  will  vary  with  the  locality,  but  probably 
in  the  majority  of  cases  it  is  not  great.  Fischer  states  that  an 
examination  of  the  street  dust  in  the  city  of  Freiburg  showed  it 
to  contain  from  five  to  seventeen  different  species  of  bacteria  in 
1  gram  of  dust  which  contained  from  24,000  to  2,000,000  organisms 
per  gram.  Graham  Smith  found  at  the  top  of  the  Clock  Tower  of 
the  House  of  Parliament  in  London  only  one-third  the  number 
of  bacteria  that  were  found  at  ground  level.  Whipple  found 
1330  bacteria  per  cubic  foot  in  air  at  street  level,  while  at  the  tenth 
story  of  the  John  Hancock  Building  in  Boston  the  air  contained 
330. 

Factors  Governing  Number  and  Kind.— The  sprinkling  of  the 
streets  greatly  increases  the  number  of  bacteria  in  the  dust,  but 
it  decreases  the  number  in  the  air.  This  is  due  to  the  fact  that 
the  moist  particles  are  not  dislodged  and  carried  into  the  air  as 
freely  as  are  the  dry. 

The  air  of  the  .country  contains  fewer  bacteria  than  does  the  air 
of  the  city.  Miquel  found  as  an  average  300  organisms  per  cubic 
meter  of  air  taken  outside  the  city  of  Paris  and  5445  bacteria  per 
cubic  meter  of  air  taken  within  the  city. 

The  number  of  bacteria  in  air  over  oceans  is  low  and  varies 
with  the  nearness  to  land.  Close  to  shore  there  are  often  very 
many,  while  at  great  distances  from  land  the  air  may  be  free  from 
bacteria. 

On  mountain  tops,  in  deserts,  and  in  other  uninhabited  regions 
the  air  is  nearly  free  from  bacteria.  The  classical  illustration  of 
this  fact  is  found  in  the  experiments  carried  on  by  Pasteur  to 
refute  the  doctrine  of  spontaneous  generation.  He  exposed  flasks 
containing  organic  infusions  in  various  localities.  Of  20  such 
flasks  exposed  to  the  air  of  Mer  de  Glace  19  showed  no  contamina- 
tion. In  similar  experiments  Tyndall  exposed  27  flasks  containing 
beef  infusion  to  the  air  of  the  Aletsch  Glacier  (8000).  None 
showed  contamination,  whereas  90  per  cent,  of  a  similar  number 
opened  in  a  hayloft  did. 

The  number  of  organisms  in  air  decreases  with  the  altitude  as 
well  as  locality.  Jean  Binot  did  not  find  a  single  microorganism 
in  100  liters  of  air  taken  on  the  summit  of  Mount  Blanc.  The 
number  rapidly  increased  on  descending. 

On  the  summit 0 

At  the  Grand  Plateau 6 

At  the  Grand  Malet 8 

At  the  Pla'ce  de  1'aiguille 14 

At  the  Mer  de  Glace 23 

At  Montanvert 49 

22 


338  BACTERIA  IN  AIR 

The  number  of  bacteria  in  the  air  varies  with  the  season,  increas- 
ing from  winter  to  summer  and  decreasing  from  summer  to  winter. 
There  is  also  a  marked  decrease  in  the  number  of  bacteria  in  the 
air  after  a  rainstorm.  The  rain  carries  them  to  the  ground  and 
also  moistens  the  surface  so  that  particles  of  dust  are  not  carried 
into  the  air  by  every  breeze.  But  the  added  moisture  of  the  soil 
greatly  increases  the  speed  of  multiplication  so  that  later  as  the 
surface  soil  dries  out  more  dust  and  with  it  a  greater  number  of 
bacteria  are  carried  into  the  air.  It  is  also  true  that  the  number 
of  microorganisms  in  the  air  decreases  in  the  winter  months  not 
because  cold  is  inimical  to  the  life  of  the  microorganisms— for  just 
the  reverse  is  true— but  the  conditions  are  not  as  good  for  them 
to  find  their  way  into  the  atmosphere.  This  is  due  to  the  fact  that 
the  soil  is  covered  with  snow  or  the  greater  moisture  prevents 
the  dust  from  being  carried  into  the  atmosphere. 

It  is  quite  evident  that  there  would  be  a  relationship  between 
the  number  of  bacteria  in  the  atmosphere  and  the  climate  of  that 
region.  Bacteria  would  multiply  rapidly  in  the  soil  of  a  warm, 
humid  district  and  these  in  turn  may  be  carried  into  the  atmosphere, 
but  the  rains  would  quickly  wash  them  out.  Hence,  there  would 
be  a  great  variation  in  a  short  time,  whereas  in  an  arid  region  the 
number  in  the  air  may  be  smaller  but  will  not  vary  as  greatly  as 
in  the  humid  region. 

The  stay  of  the  bacteria  within  the  atmosphere  will  vary,  depend- 
ing upon  a  number  of  factors: 

1.  The  hardy  spore-forming  saprophytes  may  remain  suspended 
in  the  air  for  days  or  even  weeks,,  whereas  the  frail  non-spore- 
forming  pathogens  soon  perish  due  to  either  drying  or  the  steriliz- 
ing action  of  the  sun's  rays. 

2.  Small  particles  settle  out  more  slowly  than  do  large  ones, 
for  as  the  size  of  an  object  is  decreased  the  surface  area  decreases 
less  rapidly  proportionately  than  does  the  volume.     Hence,  those 
bacteria  which  are  floating  free  in  the  atmosphere  would  subside 
more  slowly  than  those  attached  to  dust  particles. 

3.  The  time  of  suspension  is  also  determined  by  the  velocity 
of  the  air  current.     Organisms  settle  out  of  a  still  atmosphere 
more  readily  than  from  one  in  motion,  whereas  it  may  require  an 
air  current  of  considerable  velocity  to  dislodge  microorganisms 
and  bring  them  in  suspension  a  slight  current  will  sustain  them. 

4.  Moisture  in  the  atmosphere  tends  to  cause  particles  to  adhere 
together  and  as  they  grow  in  size  the  tendency  for  them  to  settle 
out  is  increased  proportionately. 

5.  Although  the  air  of  London  and  many  large  cities  contains 
numerous  particles  of  dust,  the  number  of  living  organisms  is  com- 
paratively small  as  the  various  gases  thrown  into  the  atmosphere 
have  a  slight  germicidal  effect  upon  the  bacteria. 


AIR-BORNE  INFECTION  339 

Bacteria  in  Inspired  and  Expired  Air.— Inasmuch  as  the  atmosphere 
contains  numerous  bacteria  it  is  to  be  expected  that  many  will  be 
inhaled  with  the  inspired  air.  It  is  estimated  that  a  person  living 
in  London  breathes  about  300,000  bacteria  each  day  and  individuals 
living  in  other  districts  may  take  many  times  this  number.  Most 
of  these  are  harmless  and  are  caught  on  the  moist  mucous  mem- 
branes of  the  upper  respiratory  passages,  very  few  finding  their 
way  into  the  deeper  alveoli. 

The  expired  air,  during  normal  respiration,  is  practically  free 
from  bacteria.  But  during  the  acts  of  coughing,  sneezing  and 
speaking  the  air  is  forced  out  and  with  it  bacteria,  some  of  which 
may  be  pathogens  and  if  inhaled  by  a  second  individual  may  give 
rise  to  the  specific  disease. 

Air-borne  Infection.— The  air  has  long  been  considered  as  the 
chief  vehicle  for  the  spread  of  communicable  diseases.  This 
was  but  natural,  for  until  recently  the  virus  of  these  diseases  was 
believed  to  be  gaseous  or  at  least  readily  diffusible  and  borne  by 
air  currents.  After  the  bacterial  nature  of  disease  was  discovered 
and  it  was  found  that  the  discharges  from  the  nose  and  mouth  of  the 
diseased  body  often  contains  the  causative  organisms,  and  hence 
could  readily  find  their  way  into  the  air,  this  was  a  favorite 
method  for  explaining  infection.  Recent  work,  however,  has 
demonstrated  that  the  pathogens  do  not  long  retain  their  vitality 
when  free  in  air,  and  where  infection  is  conveyed  by  air  it  is  due  to 
dust  or  droplet  infection. 

Dust  infection  occurs  only  in  the  case  of  those  diseases  caused 
by  organisms  which  can  survive  considerable  periods  of  drying. 
The  most  important  is  that  of  tuberculosis  and  is  here  confined  to 
rooms  and  dusty  places  which  have  been  occupied  by  careless 
consumptives.  The  extent  to  which  dust  is  a  factor  in  the  trans- 
mitting of  disease  is  not  well  known,  but  it  probably  is  not  great. 

Flugge  and  his  students  were  the  first  to  demonstrate  that 
minute  droplets  may  be  emitted  from  the  mouth  during  talking, 
coughing  and  sneezing.  The  droplets  may  be  carried  in  a  quiet 
room  as  far  as  twenty  or  thirty  feet.  The  large  ones  soon  settle 
out,  whereas  in  the  smaller  ones  there  is  a  great  tendency  for  many 
pathogens  to  perish.  Hence,  droplet  infection  is  conveyed  only  a 
few  feet. 


CHAPTER  XXVIII. 
WATER  BACTERIOLOGY. 

COMMON  things  are  often  little  prized,  and  this  is  true  of  water. 
Yet  there  is  no  other  compound  which  plays  so  many  and  such 
vital  parts  as  does  this  substance.  It  composes  two-thirds  of  the 
body  weight,  entering  into  the  make-up  of  every  tissue.  The 
muscles  which  do  our  work  contain  75  per  cent,  water;  the  liver 
which  acts  as  the  body  protector  against  poisons  consists  of  75 
per  cent.;  the  bones,  which  possess  a  tensile  strength  of  25,000 
pounds  per  square  inch  and  are  one  and  one-fourth  times  as  strong 
as  cast-iron,  consist  of  40  per  cent. ;  the  brain,  the  most  complicated 
and  wonderful  organ  of  the  body,  consists  of  85  to  90  per  cent.; 
the  blood,  that  cosmopolitan  fluid  which  visits  every  tissue  of  the 
body  bearing  to  it  nutrients  and  from  it  waste  products,  contains 
over  90  per  cent,  water.  All  the  secretions  of  the  digestive  glands 
consist  mainly  of  water,  and  it  is  not  there  merely  as  a  vehicle  in 
which  are  conveyed  the  active  principles,  for  it  enters  into  practi- 
cally every  chemical  reaction  through  which  carbohydrates,  fats 
and  proteins  pass  in  the  process  of  digestion  and  metabolism. 
It  is  the  fluid  in  which  are  held  the  mineral  nutrients  which  play 
such  a  vital  part  in  the  life  phenomena.  Water  gives  to  the  tissues 
their  plumpness,  carries  off  waste  products,  regulates  the  body 
temperature  and  acts  as  a  catalyzer  in  most  reactions.  Hence,  a 
substance  which  is  of  such  vital  importance  and  so  often  polluted 
or  infected  must  receive  more  than  passing  notice  by  the  bacteri- 
ologists. 

Classification  of  Waters.— From  a  bacteriological  viewpoint, 
natural  waters  are  best  classified  according  to  their  relation  to  the 
rich  layers  of  bacterial  growth  upon  the  surface  of  the  earth.  There 
are  four  distinct  classes:  (1)  Atmospheric  water,  (2)  surface  waters, 
(3)  stored  waters  and  (4)  ground  waters. 

1.  Atmospheric  water  consists  of  rain  and  snow.  It  is  really 
water  which  has  been  vaporized  and  then  condensed.  It  contains 
none  of  the  non-volatile  substances  and  should,  therefore,  more 
nearly  approach  pure  water  than  any  of  the  other  natural  sources. 
But  even  this  is  far  from  pure,  for  as  it  falls  through  the  atmosphere 
it  absorbs  gases  and  collects  large  amounts  of  floating  dirt.  Every 
one  has  observed  how  a  shower  will  wash  the  air  so  that  it  becomes 
beautifully  clean  arid  clear.  The  minute  the  water  reaches  the 


CLASSIFICATION  OF  WATERS  341 

earth  further  contamination  occurs  and  it  is  a  well-known  fact 
that  some  of  the  filthiest  water  used  for  domestic  purposes  comes 
from  rainwater  tanks.  This  is  due  both  to  the  methods  of  collect- 
ing and  of  storing  which  pollutes  but  usually  does  not  infect  it. 

2.  Surface  waters  include  rivers,  .creeks  and   smaller  streams 
and    are    immediately    exposed    to    contamination.     They    vary 
greatly  in  composition,  depending  upon  the  nature  of  the  catch- 
ment basin.     Waters  flowing  through  rock,  gravel  or  sand  forma- 
tion are  better  than  are  those  which  flow  over  or  drain  loam  or 
swamps.     But  even  the  waters  from  sand  and  gravel  regions  may 
be  polluted  or  even  infected,  depending  upon  the  relationship  borne 
by  the  drainage  basin  to  animal  life,  and  especially  to  human  beings. 
In  the  thickly  settled  portions  of  the  country  and  as  the  new  dis- 
tricts build  up  these  waters  must  be  more  carefully  protected. 
Sanitary  workers  are  being  forced  to  the  conclusion  that  it  is 
impossible  to  protect  such  waters  against  contamination,  and  as 
far  as  possible  such  waters  should  be  purified  before  they  are  used. 

3.  Stored  waters  include  lakes  and  large  ponds.    These,  when 
fresh  and  kept  free  from  the  pollution  with  the  wastes  of  human 
life  and  industry  make  admirable  sources  of  water.     On  account 
of  the  limited  area  of  the  drainage  basins  they  are  more  easily 
protected  than  large  streams.    Moreover,   the  natural  agencies 
for  purification— time,  sedimentation  and  enormous  dilution— play 
a  great  part  in  freeing  the  water  from  any  accidental  foreign  material 
which  may  find  its  way  into  the  water. 

4.  Ground  waters  are  of  two  classes:     (a)  Deep  springs  and 
wells,  from  which  most  bacteria  and  other  suspensoids  have  been 
removed  by  filtration.    Such  waters  in  passing  through  the  soil 
take  up  large  quantities  of  carbon  dioxid  which  has  been  set  free 
by  the  decay  of  organic  matter.    Water  heavily  charged  with 
carbon  dioxid  has  a  great  solvent  action  for  lime  and  other  inor- 
ganic constituents.    Hence,  while  such  waters  are  usually  safe  they 
are  hard  and  carry  large  quantities  of  organic  material.     (6)  Shal- 
low springs  and  wells  correspond  more  nearly  to  surface  waters 
and  are  often  polluted  and  at  times  infected. 

Waters  are  also  classified  as  polluted  and  infected.  A  good 
water  is  one  of  high  standard  quality,  as  determined  by  physical 
inspection,  sanitary  survey  of  the  watersheds,  clinical  experience, 
bacteriological  and  chemical  analysis. 

A  polluted  water  is  one  containing  organic  waste  of  either  animal 
or  plant  origin.  A  polluted  water  is  not  necessarily  a  dangerous 
water  but  is  always  looked  on  by  the  bacteriologist  with  suspicion. 

An  infected  water  is  one  which  contains  the  specific  micro- 
organism which  causes  disease  and  is  always  dangerous.  The 
bacteriologist  in  examining  seldom  proves  that  a  water  is  infected, 
but  draws  his  conclusions  from  indirect  evidence. 


342  WATER  BACTERIOLOGY 

Numbers  of  Bacteria  in  Waters.— The  bacterial  content  of  the 
several  waters  varies  greatly.  Atmospheric  waters  after  a  long- 
continued  storm  may  be  free  from  bacteria,  whereas  rain  after  a 
long  drought  may  contain  many.  There  is  also  a  variation  in  the 
number,  depending  upon  whether  the  rain  is  collected  in  the  country 
or  city.  Miquel  obtained  for  the  period  1883-1886  an  average 
of  4.3  bacteria  per  cubic  centimeter  in  the  country  and  19  per 
cubic  centimeter  in  Paris.  Snow  contains  rather  higher  numbers 
than  does  rain.  Janowski  found  in  freshly  fallen  snow  from  34 
to  463  bacteria  per  cubic  centimeter  of  snow-water. 

Surface  waters  are  never  free  from  bacteria,  but  the  numbers 
vary  greatly  from  a  few  hundred,  in  the  case  of  clear  mountain 
streams,  to  millions,  in  the  case  of  the  sewage  polluted  rivers. 

The  number  varies  with  the  turbidity  of  the  stream.  The 
Thames  River  carries  277  bacteria  per  cubic  centimeter  in  April, 
whereas  the  Illinois  carries  between  6000  and  8000  per  cubic  centi- 
meter. The  number  also  varies  with  the  season  of  the  year.  In 
May  the  Potomac  River  carries  about  750,  while  in  March  it 
carries  11,500  per  cubic  centimeter.  The  number  is  increased 
when  the  drainage  basin  is  manured  with  the  various  animal 
manures,  as  it  is  also  by  the  entrance  of  sewage  into  the  streams. 

The  bacterial  content  of  lakes  is  usually  lower  than  that  of 
streams,  but  shows  wide  variations.  Lake  Michigan  near  Chicago 
gives  count  for  from  68  to  2000  per  cubic  centimeter,  while  Lake 
Lucerne's  variation  is  from  8  to  51  per  cubic  centimeter. 

The  same  wide  variation  is  shown  in  ground  waters.  Shallow 
wells  and  springs  often  contain  as  many  and  just  as  dangerous 
organisms  as  do  surface  waters.  But  deep  wells  and  springs  contain 
few  organisms,  and  it  is  not  an  uncommon  experience  to  find  some 
which  are  sterile. 

The  seasonal  variation  of  bacteria  in  deep  wells  and  springs 
is  zero,  and  where  we  have  seasonal  variation  in  these  sources  of 
water  it  indicates  surface  contamination,  and  with  shallow  wells 
and  springs  it  is  often  enormous. 

Surface  waters  are  subject  to  marked  variations  in  bacterial 
contents,  especially  during  spring  and  fall,  due  to  melting  snow 
and  rains  of  these  seasons.  A  heavy  shower  is  likely  to  increase 
contamination  by  introducing  fresh  material  from  the  surface 
of  the  ground.  Prolonged  moderate  rains  may  have  the  opposite 
effect  and  after  the  main  impurities  have  been  washed  away  may 
dilute  the  stream  with  a  better  water  than  itself.  The  net  effect, 
therefore,  depends  upon  the  character  of  the  stream  as  well  as  the 
catchment  basin.  A  stream  highly  polluted  with  sewage  may 
actually  contain  fewer  bacteria  after  a  heavy  storm  than  before, 
but  a  normal  stream  contains  more,  as  emphasized  by  the  following 
data  compiled  by  Prescott  and  Winslow: 


LIGHT 


343 


MONTHLY   VARIATION  OF  BACTERIA   IN  A  NORMAL  AND   POLLUTED 

STREAM. 


July 
July 


Bacteria  per  c.c.                               Bacteria  per  c.c. 

Date,  1904. 

I 

Lahn 

Wieseck 

Date, 

Lahn 

Wieseck 

(normal)  . 

(polluted). 

1904-05. 

(normal)  . 

(polluted). 

318 

104,000 

December1 

1220 

21,200 

132 

156,800 

January1 

3668 

29,920 

ist      

840 

98,000 

February1 

5380 

11,900 

her1   

235 

28,400 

March1 

1210 

8,250 

ber1  i        420 

58,000 

April1 

4925 

5,910 

ember      ....         2340 

39,200  ' 

May 

570 

14,800 

ember1     .                            1740 

52,000 

June 

686 

50,180 

jmber       .      . 

780        ;       28,600 

Sedimentation.— Bacteria  disappear  more  rapidly  from  still  or 
slow-flowing  streams  than  from  rapid-flowing  streams,  due  to  the 
fact  that  the  transporting  power  of  a  stream  varies  as  the  sixth 
power  of  its  velocity.  A  current  moving  six  inches  a  second  will 
carry  fine  sand;  one  moving  twelve  inches  a  second  will  carry 
gravel;  four  feet  a  second,  stones  of  about  two  pounds'  weight; 
and  thirty  feet  a  second,  blocks  of  three  hundred  and  twenty  tons. 

The  sedimentation  of  bacteria  themselves  takes  place  very 
slowly  even  in  still  water,  for  the  difference  in  numbers  between 
the  top  layer  and  the  bottom  layer  of  water  in  tall  jars  in  laboratory 
experiments  of  a  few  days'  duration  is  very  slight,  being  quite 
within  the  limits  of  experimental  error.  In  the  natural  streams 
however,  the  bacteria  are,  to  a  great  extent,  attached  to  larger 
solid  particles,  and  upon  these  the  action  of  gravity  is  more  import- 
ant. Sedimentation  is  one  of  the  most  important  factors,  according 
to  Jordan,  in  purifying  waters.  He  states  that  "it  is  noteworthy 
that  all  the  instances  recorded  in  the  literature  where  a  marked 
bacterial  purification  has  been  observed  are  precisely  those  where 
the  conditions  have  been  most  favorable  for  sedimentation." 

Light.— Light  is  one  of  the  best  germicides,  for  when  it  plays 
upon  the  naked  protoplasm  of  the  bacterial  cell  it  kills  both  vege- 
tative and  spore  forms  in  a  short  time.  Opinions  vary,  however, 
as  to  the  part  played  by  light  in  destroying  bacteria  in  natural 
waters.  Buchner  found  that  plates  containing  B.  tuberculosis 
were  sterilized  in  four  and  one-half  hours  at  a  depth  of  five  feet, 
but  were  unharmed  at  a  depth  of  ten  feet.  Plates  exposed  at 
various  depths  and  containing  various  saprophytes  gave  the  fol- 
lowing counts  after  three  hours: 

1  Rain  or  high  water  due  to  previous  thaw. 


344  WATER  BACTERIOLOGY 

Before  exposure.  Sunshine.  Darkness. 

At  surface  of  water  (per  c.c.)      .      .     2100  9  3103 

Under  20  inches  of  water  (per  c.c.)     2103  10  3021 

Under  40  inches  of  water  (per  c.c.)     2140  2115  3463 

Few  studies  have  been  made  of  the  effect  of  light  on  bacteria  in 
flowing  water.  Jordan,  after  an  investigation  of  several  Illinois 
streams,  concluded  that  at  least  in  eight  moderately  turbid  waters 
the  sun's  rays  are  virtually  without  action.  Much,  therefore, 
depends  on  the  turbidity  and  speed  of  the  current,  the  maximum 
germicidal  effect  being  produced  in  shallow,  clear,  slow-moving 
water. 

Temperature.— The  action  of  temperature  upon  the  bacteria,  varies 
with  the  food  and  specific  organism.  When  they  are  in  a  medium 
in  which  they  can  grow  and  multiply,  warmth  within  reasonable 
limits  favors  their  development.  This  is  true  of  the  natural  bac- 
terial flora  and  may,  as  was  found  to  be  the  case  at  Harrisburg, 
Pennsylvania,  hold  for  B.  coli.  But  this  does  not  hold  for  the 
pathogens  which  in  the  majority  of  cases  do  not  multiply  in  water, 
and,  as  pointed  out  by  Prescott  and  Winslow,  "when  a  bacterium 
cannot  multiply,  the  only  vital  activity  which  can  take  place  is 
a  katabolic  wasting  away,  which  soon  proves  destructive,  and  the 
higher  the  temperature  the  more  rapidly  the  fatal  result  is  reached. 
A  frog  in  winter  lives  at  the  bottom  of  a  pond  breathing  only 
through  its  skin  and  eating  not  at  all,  but  as  soon  as  the  temperature 
rises  it  must  eat  and  breath  through  its  lungs  or  perish."  The 
typhoid  bacilli  will  survive  longer  in  ice  than  in  water.  The 
speed  with  which  they  perish  varies  inversely  with  the  temperature, 
as  was  found  by  Houston. 

Percentage  of  typhoid  Period  of  final 

bacilli  surviving  disappearance  of 

Temperature.  after  one  week.  bacilli,  weeks. 

0  46.00  9 

5  14.00  7 

10 0.07  5 

18 0.04  4 

In  the  natural-occurring  waters  probably  many  factors  play  a 
part;  sometimes  it  is  the  inhibiting  action  of  microorganisms  and 
their  products  on  one  another;  at  other  times  protozoa  which  feed 
upon  bacteria  and  the  development  of  which  is  directly  proportional 
to  the  temperature  of  the  medium  in  which  they  are  growing. 

Hinds  found  that  in  pure,  natural  and  distilled  water  B.  coli 
and  B.  typhosus  die  from  starvation  at  a  regular  rate.  The  rate 
of  death  increases  with  the  temperature  and  is  similar  to  the  rate 
of  a  chemical  reaction,  thus  following  the  mono-molecular  law. 

Food.— Bacteria  are  dependent  upon  food  and  respond  quickly 
to  comparatively  slight  changes  in  their  food  supply.  Wheeler 
found  that  typhoid  bacilli  would  persist  in  almost  undiminished 


CLASSES  OF  BACTERIA  345 

numbers  in  sterilized  water  from  a  polluted  well  containing  con- 
siderable organic  matter  and  kept  in  the  dark  at  20  degrees,  while 
in  purer  water  or  in  the  light  they  died  out  in  from  two  to  six 
weeks.  In  unsterilized  water  the  results  may  be  just  the  opposite, 
for  in  the  presence  of  an  abundant  supply  the  saprophytes  may 
multiply  at  the  expense  of  the  pathogens. 

Whipple  and  Mayer  find  that  the  presence  of  oxygen  is  essential 
to  the  existence  of  typhoid  and  colon  bacilli  in  water,  and  even 
small  quantities  of  acid  and  alkali  are  fatal.  It  is  for  this  reason 
that  we  find  few  organisms  in  acid  and  alkali  water  of  various 
regions.  The  factors,  therefore,  which  are  at  work  on  the  puri- 
fication of  water  are  numerous,  and  "although  it  is  hard  to  estimate 
the  exact  importance  of  each  factor,  the  general  phenomena  of  the 
self-purification  of  streams  are  easy  to  comprehend.  A  small 
brook,  immediately  after  the  entrance  of  polluting  material  from 
the  surface  of  the  ground,  contains  many  bacteria  from  a  diversity 
of  sources. 

"Gradually  those  organisms  adapted  to  life  in  the  earth  or  in  the 
bodies  of  plants  and  animals  die  out,  and  the  forms  for  which  water 
furnishes  ideal  conditions  survive  and  multiply.  It  is  no  single 
agent  which  brings  this  about,  but  that  complexity  of  little-under- 
stood conditions  which  we  call  the  environment." 

Classes  of  Bacteria.— The  bacteria  found  in  water  may  be  roughly 
classed  as:  (1)  Natural-water  bacteria,  (2)  soil  bacteria  and  (3) 
sewage  or  intestinal  bacteria.  There  is  no  hard  and  fast  line 
between  these  classes,  for  organisms  belonging  to  the  water  flora 
are  found  in  the  soil  and  water  draining  from  manured  soil  will 
contain  intestinal  organisms.  The  classification,  however,  is  valu- 
able; for  the  first  two  groups  usually  contain  the  saprophytes, 
whereas  the  third  contains  the  pathogens. 

A  number  of  attempts  have  been  made  to  classify  water  bacteria. 
Ward,  in  his  study  of  the  bacterial  flora  of  the  Thames  River, 
arranged  them  into  twenty-one  groups.  But  the  work  is  beset 
with  certain  difficulties  which  were  recognized  by  Ward,  for  he 
made  the  following  statement:  "My  work  goes  to  show  that 
species  cannot  be  made  out,  but  that  the  limits  of  the  species  are, 
in  most  cases,  far  wider  than  is  assumed  in  descriptions— in-  other 
words,  that  many  so-called  species  in  books  are  merely  variation 
forms,  whose  characters,  as  given,  are  not  constant  but  depend  on 
treatment.  How  far  this  is  true  for  any  given  case  will  have  to 
be  tested  on  the  particular  form  in  question." 

Fuller  and  Johnson,  from  a  study  of  the  bacteria  in  the  rivers 
of  America,  suggested  a  classification  containing  thirteen  groups. 
Their  system  was  based  mainly  on  morphological  data,  and  hence 
they  experienced  considerable  difficulty  in  differentiating  short 
bacilli  from  cocci. 


346  WATER  BACTERIOLOGY 

Jordan  studied  543  strains  of  bacteria  from  the  Illinois,  Missouri 
and  Mississippi  Rivers  and  grouped  them  into  the  following  classes, 
depending  upon  their  biochemical  properties: 
I.  B.  coli  communis. 
II.  B.  lactis  aerogenes. 

III.  B.  proteus. 

IV.  B.  enteritidis. 

V.  B.  fluorescens  liquefaciens. 

VI.  B.  fluorescens  non-liquefaciens. 

VII.  B.subtilis. 

VIII.  Non-gas  formers,  non-fluorescent,  non-sporeforming  bac- 
teria which  liquefy  gelatin  and  acidify  milk. 

IX.  Similar  to  Group  VIII,  save  that  milk  is  rendered  alkaline. 

X.  Similar  to  Group  VIII,  save  that  gelatin  is  liquefied. 

XL  Similar  to  Group  IX,  save  that  gelatin  is  not  liquefied. 

XII.  Similar  to  Group  XI,  save  that  the  reaction  of  milk  is  not 
altered. 

XIII.  Chromogenic  bacteria  not  included  above. 

XIV.  Chromogenic  staphylococci. 
XV.  Non-chromogenic  staphylococci. 

XVI.  Sarcinae. 

XVII.  Streptococci. 

The  natural  water  flora  are  saprophytes  and  the  most  important 
members  found  were: 

Group  V  (B.  fluorescens  liquefaciens)  is  probably*  more  often 
found  in  water  than  any  other  species.  It  liquefies  gelatin  and 
produces  a  green  fluorescence. 

Group  VI  (B.  fluorescens  non-liquefaciens)  produces  colonies  with 
a  fluorescent  shimmer  and  does  not  liquefy  gelatin.  They  are 
often  very  abundant  in  river  water. 

Group  VIII :  Organisms  which  liquefy  gelatin  and  acidify  milk. 
These  are  closely  related  to  the  proteus  group  and  some  of  them 
are  B.  liquefaciens,  B.  punctatus,  B.  circulans.  These  are  found 
more  commonly  at  some  seasons  than  at  others. 

Groups  XIII  and  XIV:  Chromogenic  bacilli  and  cocci.  The 
red-pigmented  B.  prodigiosus  belongs  to  this  type,  as  does  also  B. 
ruber,  B.  indicus,  B.  rubescens  and  B.  rubefaciens.  Those  pro- 
ducing a  yellow  or  orange  pigment  and  belonging  to  this  group  are 
B.  aquatilis,  B.  ochraceus,  B.  aurantiacus,  B.  fuhus.  At  times 
there  occur  organisms  which  produce  violet-pigment—  B.  molaceus. 
The  chromogenic  cocci  occurring  in  water  are  not  so  numerous; 
of  these,  Sarcina  lutea  is  the  most  common  species.  The  non- 
chromogenic  cocci,  which  Jordan  classes  as  Group  XV,  are  more 
numerous. 

Soil  Bacteria.— The  flood  waters  are  continually  carrying  to  the 
surface  waters  soil  organisms,  so  we  may  at  times  find  any  of 


INTESTINAL  BACTERIA  347 

the  bacteria  which  occur  in  soil  also  in  water.  Many  of  these 
find  this  an  unsuitable  medium  for  growth  and  multiplication  and 
soon  perish.  But  some  species,  among  which  are  B.  mycoides,  B. 
subtilis,  B.  megaterium  and  B.  Mesentericus  vulgatus  persist  for  a 
considerable  time. 

Intestinal  Bacteria.— These  are  usually  of  sewage  origin.  To 
this  class  belongs  a  heterogeneous  group  of  microorganisms  which 
find  their  way  into  water  from  sewage.  Many  of  them  are  true 
saprophytes  and  of  themselves  are  not  injurious,  but  their  presence 
in  a  water  constitutes  a  danger  signal  to  the  bacteriologist.  This 
is  especially  true  of  the  B.  coli  group  of  organisms,  the  natural 
habitat  of  which  is  the  intestinal  tract  of  the  higher  animal— man.- 
Hence,  whenever  there  is  opportunity  for  these  organisms  to  find 
their  way  into  waters  there  may  also  be  opportunity  for  the  patho- 
gens which  cause  typhoid  fever,  cholera  and  dysenteria  to  reach 
the  water.  It  is,  therefore,  certain  that  even  a  little  sewage  may 
cause  much  damage  if  it  enters  a  water  supply  for  only  a  few  hours 
at  rare  intervals,  but  it  is  the  slight  continuous  infections  which 
can  give  rise  to  a  prolonged  outbreak  of  disease.  It  is  well  estab- 
lished that  typhoid  bacteria  die  quite  rapidly  in  ordinary  waters, 
and  so  far  as  known  never  multiply  in  such  waters,  as  is  seen  from 
the  following  (Mills):  "To  prove  whether  typhoid-fever  germs 
would  survive  in  the  Merrimac  River  water,  when  at  the  low 
temperature  of  the  month  of  November,  long  enough  to  pass  from 
the  Lowell  sewers  to  the  service-pipes  in  Lawrence,  a  series  of 
experiments  was  made  by  the  Board  by  inoculating  water  from  the 
service-pipes  with  typhoid-fever  germs,  and  keeping  the  water 
in  a  bottle  surrounded  by  ice,  at  as  near  freezing  as  practicable, 
for  a  month  and  each  day  taking  out  one  cubic  centimeter  and 
determining  the  number  of  typhoid  germs.  The  number  continu- 
ally decreased,  but  some  survived  twenty-four  days. 
"On  the  first  day  there  were  6120  germs. 

On  the  fifth  day  there  were  3100  germs. 

On  the  tenth  day  there  were  490  germs. 

On  the  fifteenth  day  there  were  100  germs. 

On  the  twentieth  day  there  were  17  germs. 

On  the  twenty-fifth  day  there  were  0  germs." 
At  a  higher  temperature  the  life  of  the  organism  would  have 
been  of  even  shorter  duration. 

Our  information  in  regard  to  the  cholera  vibrio  is  not  quite  as 
definite,  but  experiments  indicate  that  it  may  multiply  to  some 
extent  in  sterilized  river  or  well  water,  and  that  it  maintains  its 
vitality  in  such  water  for  several  weeks  or  even  months. 

Natural  Purification  of  Water.— Nature's  methods  of  purifying 
water  are  mainly: 


348  WATER  BACTERIOLOGY 

1.  Evaporation  and   condensation  which   gives  the  purest   of 
natural  waters.    Millions  of  gallons  of  water  are  annually  evapo- 
rated from  the  surface  of  the  globe.     Thus,  we  have  an  enormous 
natural  still  by  which  water  is  constantly  being  purified  in  Nature. 

2.  The    self-purification    of    running    streams    which    although 
important  is  often  hard  to  estimate  quantitatively.     It  is  due  to 
many  factors,  chief  among  which  are:     (a)  Chemical— the  oxidation 
and  reduction  of  organic  and  inorganic  constituents  of  the  water 
with  the  formation  of  simple  substances  which  are  not-  well  suited 
to  the  maintenance  of  life  and  growth  of  many  forms  of  bacteria, 
and  the  germicidal  influence  of  sunlight  which  is  an  important  but 
very  variable  factor.     (b)    Biological— the  death  of  microorgan- 
isms through  various  not  well-understood  conditions  grouped  under 
the  heads  of  symbiosis,  antibiosis,  time  and  various  other  means. 
(c)  Physical— of  which  dilution  and  sedimentation  are  the  more 
important. 

3.  The  storage  in  lakes  and  ponds  which  through  the  prolonging 
of  the  time  of  action  greatly  intensifies  those  factors  at  work  in 
the  natural  purification  of  running  streams. 

4.  The  combined  physical,  chemical  and  biological  action  of 
soil  upon  water  which  filters  through  the  soil.     This  is  one  of 
Nature's  greatest  purifying  agents  and  stands  second  to  evapora- 
tion and  condensation  in  effectiveness. 

Artificial  Purification.— Those  methods  which  are  so  effective  in 
the  purification  of  water  under  natural  conditions  are  usually  the 
methods  which  are  made  use  of  in  the  artificial  purification  of 
water.  Only  a  few  of  the  best  known  can  be  briefly  considered 
here.  The  student  who  is  more  deeply  interested  in  the  subject 
is  referred  to  any  of  the  many  comprehensive  works  on  this  subject. 

The  slow  sand  filter  frees  water  from  impurities  through  the 
interaction  of  sedimentation,  filtration,  and  the  biological  destruc- 
tion of  organic  matter  and  bacteria.  It  has  been  extensively  used 
for  over  one  hundred  years,  but  a  great  impetus  was  given  to  this 
measure  when  Koch,  in  1893,  showed  that  the  proper  filtration  of 
the  water  from  the  Elbe  River  saved  Altona  from  an  epidemic  ot 
cholera  which  devastated  Hamburg  which  was  using  unfiltered 
water. 

The  method  consists  in  causing  water  to  pass  through  a  layer 
of  sand  of  such  fineness  and  thickness  that  the  requisite  removal 
of  suspended  substances  is  accomplished.  The  filter  as  usually 
constructed  is  a  basin  having  a  water-tight  concrete  base  on  the 
surface  of  which  are  laid  perforated  tiles  or  pipes.  These  are 
covered  with  about  a  foot  of  gravel  graded  in  size  from  25  to  3  mm. 
in  diameter  from  bottom  to  top.  Over  this  is  placed  three  or  four 
feet  of  sand  which  acts  as  the  real  filter.  The  water  passes  through 
this  and  is  conveyed  to  the  mains  by  the  underlying  pipes.  The 


CHEMICAL  METHOD  349 

suspended  material,  including  bacteria,  is  removed  by  the  sand 
which  becomes  more  efficient  as  used,  due  to  the  rapid  formation 
of  a  mat  of  finely  divided  sediment,  in  which  protozoa  often  multi- 
ply, and  assist  biologically  in  removing  many  bacteria.  In  time  the 
mat  becomes  very  thick  and  the  filtration  although  effective  is 
unduly  slow.  The  water  is  then  allowed  to  subside  below  the 
surface  and  about  half  an  inch  of  the  sand  removed,  after  which 
filtration  is  resumed.  The  sand  removed  is  washed  to  free  it  from 
collected  impurities  and  is  later  replaced  on  the  bed  after  succes- 
sive scrapings  have  reduced  the  filter  to  about  one  foot  in  thickness. 

The  filters  are  usually  divided  into  units  of  convenient  size, 
about  half  an  acre,  so  that  one  unit  may  be  cleaned  without  inter- 
ruption of  the  system.  The  slow  sand  filter  removes  about  99 
per  cent,  of  the  bacteria,  about  one-third  of  the  coloring  matter 
and  its  long  effective  use  has  established  the  fact  that  it  has  a  favor- 
able effect  upon  the  health  of  the  community  where  used. 

Chemical  Method.— The  chemical  disinfection  of  water  on  a 
large  scale  is  now  almost  exclusively  effected  with  substances 
yielding  chlorin,  chief  of  which  are  bleaching  powder  (chlorid  of 
lime),  sodium  hypochlorite  and  free  chlorin.  The  action  of  these 
substances  is  essentially  similar  and  dependent  upon  the  quantitative 
active  chlorin  which  they  contain.  They  are  usually  added  in 
quantities  sufficient  to  give  from  0.5  to  1  part  of  active  chlorin 
per  million  parts  of  water. 

The  use  of  bleaching  powder  in  the  purification  of  waters  is 
cheap,  reliable,  harmless  and  easy  of  application,  which  makes  it 
an  attractive  method,  but  when  used  on  impure  waters  containing 
organic  matter  it  gives  rise  to  amins,  chloramins  and  other  com- 
pounds of  unknown  composition  which  impart  to  the  water  unpleas- 
ant flavors. 

Alum  is  often  used  either  alone  or  in  connection  with  the  mechan- 
ical sand  filter,  and  if  used  under  controlled  conditions  is  very 
effective  and  leaves  no  undesirable  constituents  in  the  water. 
The  quantity  should  be  accurately  determined  for  each  water  as 
it  varies  with  the  turbidity  and  quantity  of  calcium  carbonate 
contained  in  the  water. 

Potassium  permanganate  is  often  used  in  the  disinfecting  of 
small  quantities  of  waters,  but  its  effectiveness  cannot  be  depended 
upon  except  against  the  cholera  spirillum.  Moreover,  the  disagree- 
able taste  and  the  color  imparted  to  the  water  are  a  serious  drawback. 

Chlorazene,  the  new  disinfectant  suggested  by  Dakin,  has  much 
which  commends  itself  for  use  in  the  disinfection  of  small  quantities 
of  water,  as  in  the  concentration  of  1  :  300,000  it  will  sterilize  ordi- 
narily heavily  contaminated  water  in  thirty  minutes.  Such  a 
concentration  imparts  a  very  slight  taste  to  the  water  but  is  per- 
fectly palatable.  It  is  non-toxic  and  if  used  for  only  short  intervals 


350  WATER  BACTERIOLOGY 

would  probably  be  without  effect  upon  the  health  of  the  individual. 
The  compound,  chlorazene  (p-sulphondichloraminobenzoic  acid— 
C^NC^SCetUCOOH),  is  excreted  in  the  urine  as  p-sulphonamido- 
benzoic  acid. 

Ice. — It  is  often  the  case  that  water  which  one  would  not  con- 
sider fit  for  drinking  is  used  in  the  manufacture  of  ice.  This 
should  not  be  the  case  as  the  freezing  of  water  reduces  only  slowly 
the  number  of  organisms  present.  In  fact  Keith  considers  that 
low  temperatures  alone  do  not  destroy  bacteria.  On  the  contrary, 
cold  appears  to  favor  longevity  doubtless  by  diminishing  destructive 
metabolism. 

Probably  the  decrease  in  number  is  due  to  mechanical  rupturing 
of  the  cell,  lack  of  oxygen,  food  and  moisture  which  are  due  to 
the  low  temperature.  Although  there  is  a  decrease  of  bacteria, 
yet  experiments  have  demonstrated  that  even  the  pathogen  EacMlus 
typhosus  may  persist  in  ice  for  one  hundred  days.  The  cholera 
vibrio  perish  much  sooner.  Hence,  the  evidence  is  conclusive 
that  just  as  pure  a  water  should  be  used  in  the  manufacture  of 
ice  as  is  required  in  domestic  supplies. 


CHAPTER  XXIX. 
WATER  AND  DISEASE. 

HISTORY  is  replete  with  facts  indicating  that  early  in  the  history 
of  the  race  there  was  a  general  conception  that  water  might  cause 
disease.  Early  tribes  sought  out  those  streams  and  springs  which 
yielded  a  generous  supply  of  cool,  clear  water.  They  followed 
them  on  their  course  to  the  sea  and  learned  that  some  furnished 
water  which  promoted  health,  whereas  the  user  of  other  waters 
suffered  certain  plagues.  Centers  of  population  sprang  up  in 
ancient  times  around  those  points  where  water  was  readily  avail- 
able and  great  expenditures  of  labor  and  treasure  were  made  to 
protect  and  carry  it  to  places  where  it  was  needed.  About  400 
B.C.  Hippocrates  pointed  out  the  danger  from  polluted  water  and 
advised  the  filtering  and  boiling  of  such  water.  But  apparently 
during  the  following  centuries  no  relationship  was  observed  between 
the  character  of  the  drinking  water  and  the  epidemics  of  typhoid, 
cholera  and  other  intestinal  diseases  which  swept  over  Europe. 
During  the  Dark  Ages  the  belief  that  water  caused  diseases  of  the 
human  race  became  very  popular.  But  the  attributing  factor  was 
thought  to  be  witches  who  by  some  occult  magic  poisoned  pure 
wells,  springs  and  streams. 

The  statements  in  the  literature  during  the  beginning  of  the 
nineteenth  century  became  more  definite,  showing  that  the  rela- 
tionship between  the  character  of  the  drinking  water  and  the 
prevalence  of  intestinal  diseases  was  being  recognized.  By  the 
middle  of  the  century  Michel  had  collected  such  a  mass  of  statis- 
tics as  to  warrant  the  conclusion  that  there  is  a  direct  relationship 
between  the  purity  of  a  drinking  water  and  typhoid  fever. 

Disease  First  Definitely  Proved  as  Due  to  Water.— The  first  clear- 
cut  demonstration  that  disease  is  caused  by  infected  water  was 
that  of  the  now  famous  Broad  Street  well  (1854)  so  ably  studied 
by  Snow.  During  this  outbreak  of  cholera  in  London  there  was 
an  enormous  concentration  of  cases  in  a  very  limited  area  just 
east  of  Regent  Street.  There  were  during  a  period  of  about  six 
weeks  over  600  fatal  cases.  A  careful  study  of  the  site,  soil,  sub- 
soil, streets,  density  and  character  of  population,  dwellings,  yards, 
closets,  cesspools,  vaults,  drains,  conditions  of  cleanliness  and 
atmospheric  conditions  revealed  nothing  of  importance.  A  study 
of  the  water  supply  revealed  the  following  facts; 


352  WATER  AND  DISEASE 

1.  Nearly  all  of  the  cases  were  nearer  a  certain  public  pump 
in  Broad  Street  than  any  other  source  of  water  and  most  of  them 
gave  a  definite  history  of  getting  water  from  the  pump. 

2.  Of  the  few  cases  which  developed  outside  of  the  area  sup- 
plied by  the  pump  most  of  them  were  known  to  have  drunk  water 
from  the  Broad  Street  well. 

3.  The  few  scattered  cases  in  distant  parts  of  London  were 
individuals  who  had  used  water  from  the  well. 

4.  Right  in  the  midst  of  the  district  was  a  workhouse  with  235 
inmates  and  a  brewery  with  70  employees,  each  having  its  own 
well,  and  there  were  only  5  deaths  in  the  workhouse  and  none  in 
the  brewery. 

5.  It  was  shown  that  a  privy  vault  and  cesspool  in  an  adjoining 
house  discharged  through  a  leaky  drain  which  ran  within  two 
feet  of  the  Broad  Street  well. 

6.  There  were  4  fatal  cases  of  cholera  in  the  house  at  the  time 
of  the  outbreak  and  earlier  cases  which  were  probably  cholera. 

It  was  not  until  1880  that  the  typhoid  bacillus  was  isolated 
by  Eberth  and  studied  in  detail  by  Gafky  in  1884  that  we  had 
definite  information  concerning  the  causative  agent  of  typhoid 
fever,  the  way  in  which  it  leaves  the  body,  and  the  routes  by  which 
it  may  reach  drinking  water.  This  same  year  Koch  isolated  the 
cholera  vibrio  from  stools  of  patients  suffering  with  the  disease. 
He  also  isolated  the  organism  from  tankwater  in  India.  We  now 
know  that  water  is  a  vehicle  for  a  number  of  infections  such  as 
typhoid  fever,  cholera,  dysentery  and  other  intestinal  diseases. 
It  may  be  the  medium  for  conveying  infections  not  now  generally 
regarded  as  water-borne.  It  may  carry  inorganic  poisons  such  as 
lead,  or  may  be  of  such  a  nature  as  to  bring  about  derangements  of 
metabolism  resulting  in  goiter,  or  may  lower  resistance,  so  as  to 
favor  infections  not  water-borne.  It  occasionally  conveys  animal 
parasites,  amebse  and  worms. 

Amount  of  Sickness  due  to  Water.— Water  is  probably  responsible 
for  more  sickness  and  death  than  any  other  article  of  diet  except 
milk.  This  is  due  to  the  facts:  (1)  That  it  is  used  raw,  while 
many  other  substances  are  rendered  sterile  by  cooking;  (2)  water 
comes  in  contact  with  numerous  substances  upon  the  earth's  surface 
and  is  a  universal  solvent;  (3)  it  is  used  as  the  great  vehicle  for  the 
removal  of  waste,  much  of  which  may  contain  pathogenic  organisms. 

It  is  difficult  to  obtain  statistics  to  indicate  accurately  the  mor- 
bidity and  mortality  due  to  impure  water,  but  Whipple  states 
that  the  average  typhoid  death-rate  in  American  cities  is  about 
35  per  100,000,  while  cities  with  a  good  water  supply  average  20. 
He,  therefore,  attributes  40  per  cent,  of  the  typhoid  fever  of  the 
United  States  to  infected  water.  Chapin,  however,  considers  it 
would  be  more  conservative  to  place  it  at  15  per  cent,  for  the  whole 
country  rather  than  at  40.  But  even  these  figures  show  a  large 


THE  MILLS-REINCKE  PHENOMENON  353 

unnecessary  mortality  and  morbidity  when  we  remember  there 
were  25,000  deaths  in  the  United  States  in  1910,  representing  at 
least  250,000  cases. 

Dysentery  and  diarrhea,  although  not  as  fatal  as  typhoid  fever 
or  cholera,  are  not  to  be  neglected,  for  when  we  consider  the  sick- 
ness and  economic  loss  resulting  each  year  in  the  United  States 
from  these  causes,  much  of  which  is  due  to  infected  water,  we 
find  that  they  are  not  negligible.  Moreover,  the  better  care  of 
drinking  water  has  resulted  in  a  marked  decrease  in  the  ravages 
of  dysentery,  for  it  is  estimated  that  the  mortality  from  dysentery 
in  England  toward  the  end  of  the  last  century  was  but  a  fraction 
of  a  per  cent,  of  what  it  was  in  the  middle  of  the  century.  More- 
over, the  reduction  of  dysentery  in  the  United  States  has  kept  pace 
with  the  advancement  made  in  water  protection  and  purification, 
as  seen  by  the  fact  that  the  death-rate  from  dysentery  in  this 
country  in  1850  was  6.32  per  cent.;  of  the  total  mortality  in  1860, 
2.65  per  cent.;  1870, 1.6  per  cent.;  and  in  1880,  less  than  1.5  per  cent. 

The  Mills-Reincke  Phenomenon.— Mills,  of  Lawrence,  Massa- 
chusetts, and  Reincke,  of  Hamburg,  Germany,  in  1893  noted 
that  the  purification  of  the  water  supplies  of  their  respective 
towns  was  followed  by  a  decline  in  the  general  death-rate  which 
was  more  rapid  than  could  possibly  be  accounted  for  by  the  death 
from  typhoid  fever.  This  condition  was  later  searchingly  studied 
by  Sedgwick  and  MacNutt  who  gave  to  it  the  name  of  the  "Mills- 
Reincke  Phenomenon."  Later  (1904)  Hazen,  a  sanitary  engineer 
formulated  a  numerical  expression  for  the  comparative  effect  of. 
purified  water  upon  the  typhoid  fever  and  total  mortality  as  fol- 
lows: "Where  one  death  from  typhoid  fever  has  been  avoided 
by  the  use  of  a  better  water,  a  certain  number  of  deaths,  probably 
two  or  three,  from  other  causes  have  been  avoided."  This  propor- 
tion varies  greatly  in  different  instances.  It  was  1  to  16  in  Ham- 
burg, in  Lawrence  1  to  4.4,  Lowell  1  to  6,  Albany  1  to  4.4  and  1 
to  1.5  in  Binghainton.  Hence,  in  all  of  the  cases  studied  by  Sedg- 
wick and  MacNutt  it  appears  to  be  sound  and  conservative,  but 
in  some  of  the  American  cities  more  recently  studied  it  does  not 
appear  so  exact. 

The  cause  of  this  decline  in  mortality  is  not  clearly  understood. 
It  may  be  due  to  the  exclusion  of  specific  pathogenic  organisms, 
to  increased  vital  resistance  resulting  from  the  use  of  a  better 
water,  or  in  some  cases  the  appearance  and  taste  of  the  water 
may  be  improved  with  the  result  that  greater  quantities  are  used, 
and  hence  a  better  condition  of  the  body  in  general.  Probably 
many  factors  are  at  work  and  these  studies  have  revealed  a  remark- 
able relationship  between  polluted  water  and  infant  mortality. 
Rosenau  considers  that  it  bids  fair  to  assume  a  causal  importance 
in  gastro-intestinal  disturbance  of  children  second  only  to  that  of 
contaminated  milk. 
23 


354 


WATER  AND  DISEASE 


Cholera.— Water  has  been  proved  to  be  the  causative  agent  in 
the  conveying  of  cholera  in  a  number  of  instances.  The  two  best 
known  cases  are  that  of  the  Broad  Street  well,  which  has 
already  been  considered,  and  the  epidemic  of  1892  in  Hamburg. 
This  latter  will  ever  remain  classic  on  account  of  the  clearness  of 
the  circumstances  and  the  fact  that  there  is  no  missing  link  in 
the  chain  of  evidence,  as  the  cholera  vibrio  was  isolated  from  the 
Elbe  River  water. 

The  Hamburg  epidemic  occurred  in  1892,  and  in  a  little  over 
two  months  there  were  17,000  cases  with  8605  deaths,  whereas 
Altona,  which  in  reality  forms  with  Hamburg  one  large  city,  was 
practically  free.  The  two  cities  are  built  on  the  same  soil,  pro- 
vided with  the  same  sewage' system,  and  have  the  same  climatic 
conditions.  They  have  the  same  social  customs  and  were  sepa- 
rated only  by  a  political  boundary  line.  The  boundary  runs 
through  a  street  on  one  side  of  which  is  Altona  and  on  the  other 
Hamburg.  They  have  separate  water  supplies,  but  both  derive 
their  water  from  the  Elbe  River  which  is  a  grossly  polluted  stream. 
However,  the  water  supply  for  the  city  of  Altona  was  purified  by 
filtration,  while  that  of  Hamburg  was  not.  The  boundary  of 
the  epidemic  was  just  as  clear  as  was  that  of  the  water  system,  or 
in  the  words  of  Koch,  "cholera  in  Hamburg  went  right  up  to  the 
boundary  of  Altona  and  there  stopped.  In  one  street,  which  for 
a  long  way  forms  the  boundary  there  was  cholera  on  the  Hamburg 
side,  whereas  the  Altona  side  was  free  from  it." 

Typhoid.— Contaminated  water  was  the  first  recognized  and 
probably  the  most  significant  vehicle  of  typhoid  infection.  The 
improvement  in  water  supplies  during  recent  years  has  been  respon- 
sible for  the  reduction  in  typhoid  morbidity.  The  results  compiled 
by  Kober  clearly  show  the  effect  of  improved  water  supplies  on 
typhoid  mortality  in  American  cities. 

EFFECT   OF  WATER  PURIFICATION   ON   GENERAL  AND  TYPHOID 
DEATH-RATE. 


City. 

General 
death-rate 
before 
change  of 

Same 
after. 

Percentage 
reduction. 

Typhoid 
fever 
death-rate 
before 

-•L                      e 

Same 
after. 

Percentage 
reduction  . 

water 

cnange  01 

supply. 

water 

supply. 

Providence,  R.  I.     . 

19.3 

19.0 

+    1.6 

21.8 

13.7 

+  37.2 

St.  Louis,  Mo.    .      .        18.0 

16.1 

+  10.6 

39.2 

19.1 

+51.3 

Youngstown,  O.       .  j      15.6 

15.1 

+  3.2 

96.1 

39.1 

+  59.4 

Ithaca,  N.  Y.     .      . 

16.4 

15.1 

+  7.9 

108.8 

25.3 

+  76.8 

Paducah,  Ky.     . 

23.4 

17.4 

+23.0 

82.1 

78.7 

+   4.2 

Watertown,  N.  Y.  . 

15.5 

17.2 

—11.1 

100.6 

38.2 

+  62.1 

Paterson,  N.  J.  .      .        17.2 

16.5 

4.  1 

28.2 

11.9 

+  57.8 

Binghamton,  N.  Y.  !       17.6 

17.6 

0 

40.8 

13.4 

+  67.2 

1                     !                      ! 

TYPHOID  355 

Water  still  remains  the  most  important  single  channel  by  which 
the  typhoid  bacilli  reach  the  human  body.  Estimates  vary  as  to 
the  actual  percentage  of  typhoid  cases  which  are  referable  to  water 
infection.  It  is  placed  by  various  authors  at  from  10  to  40  per 
cent.  According  to  Gay,  Schuder  found  that  of  640  typhoid 
epidemics  22  per  cent,  were  due  to  water.  Schegehdahl  found 
that  of  682  cases  about  33  per  cent,  were  water-borne.  Typhoid 
is,  therefore,  the  most  important  water-borne  disease. 

The  proof  that  a  typhoid  epidemic  is  due  to  water  infection 
is  usually  indirect,  for  the  actual  isolation  of  the  offending  organ- 
ism is  effected  with  considerable  difficulty  and  has  been  accom- 
plished in  only  seven  or  eight  cases.  However,  in  those  cases 
where  it  is  found  it  is  not  always  possible  to  prove  that  it  was 
present  at  the  time  the  infection  occurred.  Strong  presumptive 
evidence  is  given  whenever  waters  are  proved  through  the  presence 
of  colon  bacillus  to  have  been  infected  by  sewage. 

The  best  evidence,  however,  obtainable  that  a  specific  typhoid 
outbreak  is  due  to  polluted  water  is  that  obtained  by  the  epidemi- 
ologist. He  knows  that  the  important  characteristics  of  water- 
borne  epidemics  are: 

1.  They  may  be  preceded  by  a  period  of  dysentery. 

2.  The  epidemic  usually  has  a  sharp  onset,  the  curve  rising  to  a 
peak  and  the  decline  being  rapid. 

•     3.  The  cases  are  quite  evenly  divided  over  the  city,  that  is, 
provided  the  city  is  served  by  a  municipal  supply. 

4.  They  nearly  always  occur  in  the  spring,  fall,  or  winter. 

5.  The  pollution  is  usually  nearby  and  the  epidemic  is  of  short 
duration  unless  there  be  a  continuous  source  of  new  infecting 
material. 

The  work  of  the  epidemiologist  is  vividly  portrayed  by  Hill 
as  follows: 

"To  illustrate  the  general  principles,  let  us  suppose  notification 
be  received  that  a  typhoid  fever  outbreak  exists  in  a  far-off  com- 
munity. The  public  health  detective  packs  his  grip  and  goes. 
He  knows  no  details;  he  has  never  heard  of  this  particular  com- 
munity before;  he  has  not  even  any  general  information  about  the 
character  of  the  country;  he  enters  the  community  with  no  pre- 
conceived ideas.  But  he  does  know  how  typhoid  fever  originates 
and  how  it  spreads.  Water,  milk,  food,  flies  and  fingers  are  the 
routes— typhoid  cases  or  typhoid  carriers,  the  source.  His  duties 
are  to  find  both;  and  to  find  them,  not  as  a  scientific  amusement, 
or  as  a  matter  of  record;  not  to  furnish  food  for  speculation— above 
all  not  to  make  a  show  of  doing  something— but  to  stop  the  outbreak, 
and  then  to  advise  measures  to  prevent  recurrence. 

"The  public  health  detective  on  entering  the  community  affected 
by  typhoid  fever  does  not  first  examine  the  water-supply,  the 


356  WATER  AND  DISEASE 

milk  supply,  the  sewage  disposal  system,  the  markets,  the  back 
alleys,  the  dairies,  or  anything  else.  He  goes  directly  to  the  bedsides 
of  the  patients.  Of  course  he  must  obtain  the  names  and  addresses 
of  the  patients  from  someone— from  the  local  health  officer,  if  he 
has  them;  from  the  attending  physician,  if  the  health  officer  has 
no  list;  from  the  lay  citizens  themselves,  if  no  one  else  is  immediately 
available.  The  more  complete  the  list,  the  faster  he  can  work, 
because  then  he  is  not  compelled  to  hunt  up  the  cases  personally. 
But  if  there  be  no  list,  he  begins  making  one  himself.  His  inten- 
tion is  to  see  just  as  many  patients  as  he  can,  for  each  furnishes  evi- 
dence and  he  wants  it  all.  But  he  knows  that  it  is  not  always 
necessary  at  this  stage  to  see  absolutely  all  the  patients,  so  long 
as  he  sees  the  majority. 

"Reaching  the  patient's  bedside,  his  investigation  begins. 
Automatically,  almost  mechanically,  he  decides  whether  or  not 
the  patient  has  typhoid  fever  or  not.  Satisfied  on  that  point, 
his  first  question  is  not,  'Tell  me  all  the  different  water  supplies 
you  have  used,  or  all  the  sources  of  milk  you  have  used.'  The 
first  question  is,  '  When  did  you  first  show  the  earliest  symptoms  of 
the  disease?'  Why?  Because  this  date  once  fixed,  at  which  infec- 
tion entered  the  patient's  mouth  is  fixed  also,  i.  e.,  a  date  between 
one  and  three  weeks  previous  to  the  date  of  the  earliest  symptoms. 
Remember  that  at  that  .stage  the  detective  may  not  have  even  an 
inkling  as  to  which  of  the  usual  factors— water,  milk,  food,  flies 
or  fingers— is  involved.  Still  less  can  he  guess  which  particular 
water  supply,  milk  supply,  etc.,  of  the  many  possible  ones,  may  be 
the  guilty  one.  But  the  answer  to  this  question  reduces  possi- 
ble routes  to  those  used  by  this  patient— not  at  any  time— but  during 
a  specific  period,  i.  e.,  from  one  to  three  weeks  preceding  his  date 
of  earliest  symptoms. 

"Not  yet,  however,  are  the  milk  and  water  questions  offered. 
The  second  question  is  'Where  were  you  during  that  period?' 
Why?  Because  if  the  patient  were  not  in  the  community  during 
that  period,  he  could  not  have  contracted  his  infection  within  it, 
and  does  not  belong  to  the  outbreak  under  examination  at  all  but 
to  some  other.  He  is  in  brief  an  'imported  case,'  and  while,  of 
course,  he  is  to  be  supervised  lest  he  spread  his  infection  to  others, 
he  cannot  help  to  locate  the  source  of  the  main  outbreak— unless 
perchance  he  be  himself  that  source,  i.  e.,  the  introducer  to  the 
community  of  the  original  infection.  If  he  be  an  imported  case 
he  is  noted  for  further  reference  and  the  detective  goes  to  another 
patient.  If  not,  the  questions  continue.  But  not  yet  is  water 
or  milk  or  flies  mentioned.  The  third  question  is,  'Were  you  asso- 
ciated during  your  period  of  infection  with  any  then  known  typhoid 
case?'  Why?  Because  such  association,  especially  if  intimate, 
makes  it  more  probable  that  the  case  under  examination  received 


TYPHOID  357 

his  infection  from  the  preceding  case,  rather  than  from  any  general 
route  and  that  he  is,  therefore,  a  'secondary  case.'  If  he  had 
such  associations,  this  is  noted  for  further  reference  and  the  investi- 
gator passes  on  to  another  bedside.  If  not,  the  questions  continue, 
and  now  at  last  take  up  milk,  water,  food,  etc.,  but  of  course  only 
so  far  as  to  determine  those  used  by  the  patient  during  his  infec- 
tion period. 

"Then  the  investigator  passes  to  the  next  patient.  What  has 
he  learned  so  far?  Nothing  much  yet.  But  he  has  narrowed  the 
possible  routes  of  infection  to  certain  water  supplies,  certain  milk 
supplies,  certain  food  supplies,  etc.,  i.  e.,  those  used  by  the  first 
patient  during  a  certain  period,  and  he  has  done  this  in  thirty  minutes 
—in  scarcely  the  time  it  takes  for  the  old-style  investigator  to  get 
his  bottles  ready  to  collect  his  first  water  sample. 

"At  the  bedside  of  the  second  patient,  the  same  inquiries  in 
the  same  order  are  made.  If  this  second  patient  be  an  imported 
case,  or  a  secondary  case,  he  also  is  merely  noted  for  future  refer- 
ence. If  he  be  a  primary,  however,  the  origin  of  his  drinking  water, 
milk,  food,  etc.,  during  his  infection  period  are  also  ascertained. 
Perhaps  he  coincides  with  the  first  patient  in  every  detail  of  aliment- 
ary supplies,  in  history  and  associations.  If  so,  nothing  much  has 
been  added  to  the  detective's  knowledge.  But  more  than  likely, 
dissimilarities  have  developed.  Since  the  responsible  water  supply, 
milk  supply,  etc.,  must  be  one  of  those  water  supplies,  milk  supplies, 
etc.,  used  in  common  by  primary  cases,  all  those  not  common  to 
both  of  these  primary  cases  may  be  dropped  from  consideration 
(except  in  rare  instances  of  multiple  routes).  Thus,  if  both  have 
used  the  same  water,  water  from  that  origin  remains  as  a  possi- 
bility. But  if  the  water  supplies  have  been  different,  water  is 
eliminated  from  the  question  entirely.  If  the  milk  supplies  are 
identical,  milk  remains  as  a  possible  route  of  infection;  if  not, 
milk  is  eliminated  from  the  question  entirely. 

In  brief,  provided  the  information  obtained  be  reliable,  and  it 
is  part  of  the  public  health  detective's  training  to  distinguish  at 
a  glance  truth  from  falsehood,  the  honestly  mistaken,  or  forgetful, 
or  stupid  replies  from  the  reliable  ones— and  above  all  never  to 
believe  anything  (to  the  extent  of  recording  it)  unless  it  is  checked, 
confirmed  and  established  as  a  fact,  the  modern  investigator  has 
in  one  hour  narrowed  his  investigation  to  a  point  which  the  old- 
style  investigator  often  would  not  reach  for  weeks. 

"And  so  from  patient  to  patient  the  inquiry  proceeds.  In  the 
course  of  the  day  the  investigator  has  seen  perhaps  30  patients. 
The  tabulation  (probably  already  made  in  his  own  mind)  shows, 
say,  3  imported  cases,  5  secondaries,  2  uncertain  or  indefinite. 
The  remaining  primary  cases  show  in  common,  say,  1  water  supply 
only,  the  milk,  etc.,  varying;  or  1  milk  supply  only,  the  water, 


358  WATER  AND  DISEASE 

etc.,  varying;  or  no  connection  except  attendance  at  some  one 
social  function. 

"Going  straight  to  the  route  thus  indicated,  the  public  health 
detective  quickly  confirms  the  indications  of  his  results.  He  knows 
that  the  route  indicated  must  be  the  guilty  one,  for  only  that 
route  can  account  for  all  the  cases.  He  concentrates  on  that 
route  until  the  evidence  is  complete— when  and  how  that  route 
became  infected,  when  and  by  what  sub-routes  the  infection  was 
distributed,  why  it  infected  the  patients  found  and  not  others,  etc. 

"In  this  illustration  I  have  assumed  complete  ignorance  on  the 
part  of  the  epidemiologist  as  to  everything  connected  with  the 
community  he  is  investigating,  except  what  he  finds  by  cross-exam- 
ining the  patients.  As  a  matter  of  fact,  every  epidemiologist, 
however  much  a  stranger  to  the  particular  community  he  enters, 
begins  to  learn  about  it  from  the  moment  he  enters  it. 

"Thus,  almost  unconsciously  he  notes  the  size  of  the  town  and 
compares  it  with  the  number  of  cases  reported  as  existing;  if  it 
is  summer  time  he  almost  automatically  notes  the  presence  or 
absence  of  open  toilets  in  the  backyards,  of  manure  piles  and  of 
garbage  cans— all  bearing  upon  fly  infection.  If  it  is  winter  time 
or  the  community  be  well  sewered,  he  does  not  even  consider  flies. 
If  the  cases  are  grouped  in  one  quarter  of  the  town,  while  the  public 
water  supply  extends  all  over  it,  he  tentatively  eliminates  the 
water  supply  before  he  asks  a  question.  If  good  surface  drainage 
and  a  sandy  soil  exist,  or  driven  wells  are  chiefly  in  vogue,  he 
tentatively  eliminates  well  water— even  before  he  registers  at  the 
hotel. 

"This  is  not  and  cannot  be  a  complete  synopsis  of  all  the  com- 
binations of  circumstances  which  the  epidemiologist  meets.  It  is 
intended  to  illustrate  his  methods  and  to  show  why  they  are  incred- 
ibly rapid  and  incredibly  accurate— how  they  eliminate  specula- 
tion and  guarantee  a  correct  solution— which  means,  of  course,  the 
achievement  of  the  great  end,  the  finding  of  proper  measures  for 
suppression. 

"As  soon  as  the  route  is  indicated,  he  must  go  to  that  route,  and 
establish  beyond  peradventure  that  it  was  in  truth  responsible. 
A  water  supply  cannot  convey  typhoid  if  typhoid  fever  discharges 
have  not  entered  it.  There  is  no  object  in  attributing  an  outbreak 
to  fly  infection  from  toilets  into  which  typhoid  feces  have  not 
been  discharged  at  such  a  time  as  to  account  for  the  cases.  A 
milk  supply,  not  handled  at  some  point  by  an  infected  person,  nor 
adulterated  at  some  time  with  infected  extraneous  matter  cannot 
convey  typhoid.  Whatever  his  results,  they  cannot  be  true  unless 
they  are  consistent— they  should  not  be  accepted  unless  they  are 
provable — and  proved. 

"If  the  public  health  detective  is  familiar  with  the  communitv 


TYPHOID  359 

where  the  outbreak  occurs,  including  its  water  supplies,  its  milk 
supplies,  the  sociological  relationships  of  its  people,  etc.,  he  can 
often  tentatively  determine  the  cause  of  the  outbreak  by  a  mere 
inspection  of  the  names  and  addresses  of  primary  cases,  especially 
if  plotted  on  a  map  of  the  community,  taking  into  account  also 
the  time  of  year,  and  other  general  points.  But  such  deductions, 
while  often  wonderfully  reliable,  can  never  be  as  conclusive  and 
satisfactory  as  are  the  results  of  an  investigation  by  even  a  total 
stranger,  if  the  investigation  be  conducted  as  above  described/' 

REFERENCES. 

Savage,  W.  G.:     The  Bacteriological  Examination  of  Water  Supplies. 

Harracks,  W.  H.:  An  Introduction  to  the  Bacteriological  Examination  of  Water. 

Prescott  and  Winslow:     Elements  of  Water  Bacteriology. 

Thresh:     The  Examination  of  Water 'and  Water  Supplies. 

Mason:     Water  Supplies. 

Rosenau:     Preventative  Medicine  and  Hygiene. 

Don  and  Chisholm:     Modern  Methods  of  Water  Purification. 


CHAPTER  XXX. 
SEWAGE  AND  SEWAGE  DISPOSAL. 

MAN  early  learned  that  both  esthetic  and  sanitary  reasons 
demand  that  sewage  be  properly  treated.  In  the  early  history 
of  the  race  and  also  of  a  district  the  pit  or  trench  was  used  for  the 
disposal  of  the  refuse.  Later  this  was  lined  with  stone,  brick 
or  cement  to  partly  prevent  diffusion  into  the  surrounding  soil, 
and  hence  the  contamination  of  the  well.  This,  when  properly 
covered,  became  the  cesspool  which  is  largely  in  use  in  the  rural 
districts  today.  As  population  increased  with  its  constantly 
growing  volume  of  human  waste  the  old  methods  became  inade- 
quate, and  hence  there  has  developed  the  modern  sewage  system. 

Source,  Composition  and  Quantity  of  Sewage.— A  city's  sewage 
consists  of  the  public  water  supply  carrying  human  and  animal 
'excreta,  refuse  from  the  kitchen,  laundry,  manufacturing  estab- 
lishments and  the  dust  and  dirt  of  the  streets.  Its  quantity  is 
directly  proportional  to  the  consumption  of  water  in  the  district. 
In  small  cities  it  may  be  as  low  as  forty  or  fifty  gallons  per  capita 
daily,  whereas  in  larger  cities  it  may  reach  from  100  to  200  gallons 
or  over. 

Its  composition  depends  upon  the  density  of  population,  the 
number  and  kinds  of  manufacturing  establishments,  and  whether 
there  is  a  separate  or  combined  system.  Where  the  combined 
system  is  used  the  composition  and  quantity  of  the  sewage  varies 
with  the  rainfall  and  street  washing.  There  is  also  a  diminution 
in  quantity  and  composition  at  night. 

Fuller  gives  the  estimated  amount  of  dry  suspended  solids  in 
the  New  York  City  sewage  per  1000  inhabitants  annually  as 
follows: 

Tons  per  1000 

Inhabitants 
Material.  annually. 

Feces .      .  14 

Toilet  paper  and  newspaper 8 

Soap  and  washings 11 

Street  wastes 8 

Miscellaneous 4 

Total       ...."............     45 

From  the  viewpoint  of  purification  sewage  contains  proteins, 
carbohydrates,  fats,  soaps,  urea  and  other  organic  substances. 


BACTERIA  IN  SEWAGE  361 

The  important  elements  *  present  are  nitrogen  and  sulphur.  The 
quantity  of  these  present  determines  the  nature  and  repulsiveness 
of  the  resulting  products. 

Bacteria  in  Sewage.— The  number  and  kind  of  bacteria  in  sewage 
varies  widely  with  its  composition  and  origin.  According  to  Fuller 
it  contains  320  billion  for  each  person  connected  with  the  sewer 
system.  Johnson  found  B.  coli  to  average  about  500,000  per  c.c. 
He  isolated  the  following  species  from  the  crude  sewage  of  Columbus : 

Number  of  times 
Species.  found. 

B.  liquefaciens 21 

B.  coli  communis 19 

B.  liquidus 8 

B.  mesentericus  vulgatus 7 

B.  bruneus 4 

B.  hyalinus   . 3 

B.  fuscus 3 

B.  delicatulus -3 

B.  pyocyaneus 2 

B.  fluorescens 2 

B.  circulans 2 

B.  nibilus 2 

B.  weichselbaumii 2 

B.  sporogenes 

B.  stellatus 

B.  helvolus 

B.  cereus 

B.  cloaccB 

B.  proteus  zenkeri 

B.  monadiformis 

B.  aeris  muintissimus 

M .  tetragenus  mobilis  ventriculi 

M .  casei 

M.  albicans  amplus 

M.fervidosus 

Str.  coli  gracilis 

Sir.  enteritis 

Sarcina  alba 

Ps.  turcosa 

Ps.  nebulosa 

Ps.  ochracea 

In  addition  to  these,  many  of  the  pathogens  may  find  their  way 
into  sewage  and  survive  for  various  lengths  of  time. 

However,  the  interest  centers  more  in  the  changes  produced  by 
the  various  bacteria  found  in  sewage  than  in  the  specific  classes. 
Most  of  them  are  not  only  harmless,  but  of  genuine  importance 
in  the  economy  of  Nature  through  the  scavengering  work  which 
they  accomplish.  A  few  of  them  are  dangerous  on  account  of 
their  causing  certain  infectious  diseases.  Many  of  them  play 
an  important  role  in  decomposing  sewage  with  the  formation  of 
malodorous  gases  and  products  associated  with  putrefactive 
nuisances. 

The  modern  tendency  is,  therefore,  to  classify  sewage  bacteria 


362 


SEWAGE  AND  SEWAGE  DISPOSAL 


from  a  physiological  viewpoint.     They  may  roughly  be  divided 
into  four  classes— hydrolyzing,  oxidizing,  reducing  and  pathogenic. 
Fuller  gives  the  estimated  constituents  of  average  sewage  as 
follows: 


Grams  per  capita 
daily. 

Parts  per 
million. 

Oxygen  consumed: 
Two  minutes'  boiling      
Five  minutes'  boiling 

15.0 
22.0 

39.6 
58.0 

Nitrogen: 
Free  ammonia 
Albuminoid  ammonia 
Organic     

Total       .      . 

7.0 
2.5 
8.0 

17.5 

18.5 
6.6 
21.1 

46.2 

Chlorin    

19.0 

50.2 

Fats  

19.0 

50.2 

Dissolved  matter: 
Mineral     
Organic  and  volatile       ...                  . 

58.0 
40.0 

140.0 
106.0 

Total       

98.0 

246.0 

Total  solids: 

152  0 

402.0 

Organic  and  volatile        

77.0 

203.0 

Total       ............ 

229.0 

605.0 

Bacteria,  322  billion  per  capita  daily. 

Hydrolyzing  Bacteria.— Probably  most  of  the  early  changes  which 
occur  are  hydrolytic,  that  is,  the  substance  is  caused  to  take  up 
water,  becomes  unstable,  and  for  some  reason  falls  into  fragments, 
thus  often  passing  from  a  non-soluble  compound  of  complex  con- 
stitution to  a  simple  soluble  substance. 

Protein  liquefaction  belongs  to  this  type  of  changes  and  is  brought 
about  by  a  great  variety  of  bacteria  working  in  conjunction  with 
each  other.  The  proteins  are  hydrolyzed  by  successive  stages  to 
proteoses,  peptones,  peptids,  amino-acids  and  finally  to  ammonia, 
carbon  dioxid,  methane,  hydrogen,  etc.  It  probably  corresponds 
in  the  main  with  the  changes  which  have  been  considered  under 
ammonification.  The  final  products  vary  widely,  depending  upon 
whether  the  process  is  being  carried  on  under  aerobic  or  anaerobic 
conditions.  The  tendency  is  for  it  to  partake  more  of  putrefaction 
.in  the  septic  tank  and  decay  in  soil. 


REDUCING  BACTERIA  363 

Cellulose  fermentation,  next  to  protein  hydrolysis,  is  the  most 
important  work  of  bacteria  in  sewage  purification.  Paper,  cotton 
fabric,  wood  and  other  cellulose-containing  substances  are  rapidly 
attacked  by  various  organisms  with  the  production  of  soluble 
substances— starches,  sugars,  acids  and  finally  carbon  dioxid, 
methane  and  hydrogen. 

Probably  fewer  organisms  possess  the  power  of  saponifying  fat 
than  of  liquefying  proteins  or  hydrolyzing  cellulose.  For  this 
reason  and  also  due  to  the  fact  that  the  fat  tends  to  rise  to  the 
surface  out  of  the  sphere  of  bacterial  action,  there  is  a  great  ten- 
dency for  the  fat  to  accumulate.  At  times  this  may  accumulate 
around  some  solid  and  give  rise  to  "grease  balls"  which  cause 
clogging  of  pipes.  The  fat  which  is  acted  upon  by  bacteria  is 
broken  into  fatty  acids  and  glycerin.  The  fatty  acids  are  quite 
resistant  to  further  bacterial  activity,  but  the  glycerin  is  rapidly 
broken  into  simpler  products. 

Oxidizing  Bacteria.— The  complex  microflora  of  the  sewage  must 
have  energy.  This  they  get  in  a  great  degree  from  the  oxidation 
of  the  comparatively  simple  products  yielded  through  the  hydrolysis 
of  the  proteins,  carbohydrates  and  fats.  These  are  changed  prob- 
ably similarly  to  the  acetic  acid  fermentation  with  the  production 
of  acids  and  finally  carbon  dioxid  and  water. 

The  ammonia  liberated  through  the  deaminization  of  the  amino- 
acids  is  oxidized  by  the  Nitrosomonas  to  nitrous  acid  and  by  the 
Nitromonas  to  nitric  acid. 

Reducing  Bacteria.— The  nitrites  and  nitrates  formed  by  the 
nitrifying  bacteria  are  in  a  great  measure  reduced  to  free  nitro- 
gen through  denitrification.  The  sulphur  in  the  protein  molecule  is 
liberated  as  sulphates,  sulphur  dioxid  and  hydrogen  sulphid.  The 
sulphate  formed  is  reduced  to  hydrogen  sulphid.  This  reacts  with 
the  small  amounts  of  iron  and  other  metals  present  with  the  result- 
ing black  residue  of  metallic  sulphids  always  found  on  the  bottoms 
of  tanks  and  streams  in  which  sewage  is  decomposing. 

Each  of  these  processes  is  going  on  simultaneously  in  sewage 
and  the  one  is  dependent  upon  the  other,  there  being  a  true  bio- 
logical cycle,  as  is  pointed  out  by  Whipple. 

"The  decomposition  and  oxidation  of  the  organic  matter  in 
sewage  are  brought  about  by  bacteria,  and  the  bacteria  serve  as 
food  for  protozoa  and  other  forms  of  microscopic  animal  life.  The 
dissolved  organic  matter  in  sewage  serves  as  food  for  algse.  These 
algae  and  protozoa  are,  in  turn,  consumed  by  rotifers  and  Crustacea, 
while  the  latter  form  the  basis  of  food  supply  for  various  aquatic 
animals  and  fishes.  Thus,  there  is  a  continuous  biological  cycle. 
Again,  animal  forms  require  oxygen  and  produce  carbonic  acid, 
while  plants  consume  carbonic  acid  and  produce  oxygen.  Where 
these  processes  occur  normally  and  with  a  proper  equilibrium  main- 


364  SEWAGE  AND  SEWAGE  DISPOSAL 

tamed  between  animal  and  plant  life,  offensive  conditions  do  not 
result,  but  where  abnormal  conditions  are  produced,  as,  for  example, 
by  the  discharge  of  excessive  quantities  of  sewage  or  trade  wastes 
into  a  stream,  a  depletion  of  the  dissolved  oxygen  may  follow,  or 
there  may  be  an  overproduction  of  algae  so  that  the  conditions 
become  offensive.  It  is  coming  to  be  realized  that  in  order  to 
properly  determine  the  dilution  required  in  any  particular  case 
the  conditions  required  to  bring  about  this  condition  of  biological 
equilibrium  must  be  determined." 

Pathogenic  Bacteria.— Owing  to  the  origin  and  nature  of  sewage 
it  may  at  any  time  contain  pathogenic  bacteria.  The  ones  with 
which  the  sanitarian  is  most  concerned  are  the  typhoid,  cholera 
and  dysenteria,  but  it  is  always  possible  for  many  other  species 
to  find  their  way  into  sewage  and  from  it  cause  infection.  This 
is  especially  true  of  B.  tuberculosis  which  is  quite  resistant  to  putre- 
faction. With  the  exception  of  the  cholera  and  dysentery  organ- 
isms, there  is  no  evidence  that  they  ever  multiply  in  sewage,  and 
they  produce  no  appreciable  change  in  its  composition. 

The  majority  of  the  pathogens  soon  die  in  sewage.  The  results 
as  reported  by  Whipple  for  typhoid  bacilli  are  given  in  Fig.  44. 

The  speed  with  which  the  typhoid  bacilli  disappear  from  water 
varies  with  the  vitality  of  the  organism,  the  temperature  of  the 
water,  the  organic  matter,  and  the  bacterial  antagonism  exerted 
by  other  organisms.  Typhoid  bacilli  seem  to  die  more  quickly  in 
sewage  than  in  fairly  pure  water,  probably  because  of  the  great 
bacterial  antagonism  existing.  Furthermore,  the  absence  of 
oxygen  probably  plays  an  important  part,  as  Whipple  found  oxygen 
necessary  for  longevity  of  typhoid  bacilli.  Jordan  thus  summarizes 
our  present  knowledge  of  the  longevity  of  typhoid  bacilli: 

"Laboratory  experiments  have  shown  that  the  typhoid  bacillus 
can  survive  in  sterile  water  in  glass  vessels  for  upward  of  three 
months,  and  for  possibly  two  or  three  weeks  in  unsterilized  ground 
or  surface  water.  Other  evidence  indicates  that  the  bacillus  is 
able  to  travel  in  water  a  distance  of  at  least  140  kms.,  and  to  retain 
its  vitality  in  natural  bodies  of  water  for  at  least  four  or  five  days. 
It  is  possible  that  water  may  continue  to  be  the  vehicle  of  infection 
during  a  much  longer  period,  but  the  available  data  point  to  a 
comparatively  short  duration  of  life  of  the  specific  germ  in  the 
water  of  flowing  streams.  Under  ordinary  conditions  no  multi- 
plication of  the  typhoid  bacillus  takes  place  in  water,  even  when 
a  considerable  amount  of  organic  matter  is  present,  but,  on  the 
contrary,  a  steady  decline  in  numbers  goes  on.  The  history  of 
typhoid  epidemics  tends  to  show  that  sewage  pollution  is  to  be 
feared  chiefly  when  the  sewage  is  fresh,  and  that  the  danger  of 
infection  diminishes  progressively  with  the  lapse  of  time. 

"In  soil  in  the  fecal  matter  of  privy  vaults  the  duration  of  life 


PATHOGENIC  BACTERIA 


365 


of  the  typhoid  bacillus  is  much  longer  than  in  water.  Levy  and 
Kayser  found  typhoid  bacilli  in  soil  that  had  been  manured  fourteen 
days  previously  with  the  five-months-old  contents  of  a  vault.  The 
evidence  that  any  genuine  multiplication  can  take  place  in  the 


IVNIOiaO  JO  J.N30U3d 


soil  is  not  convincing,  but  it  has  been  proved  that  the  bacillus 
may  be  carried  by  water-currents  to  a  considerable  distance  from 
the  point  where  it  was  first  introduced.  Infection  of  wells  and 
small  water-courses  is  thus  brought  about  sometimes  by  the  wash- 


360  SEWAGE  AND  SEWAGE  DISPOSAL 

ing  of  bacilli  out  of  soil  in  which  they  may  have  lain  dormant  for 
many  months.  The  persistence  of  typhoid  fever  around  certain 
habitations  may  be  plausibly  explained  on  the  supposition  of  an 
extensive  soil  infection.  There  is  no  doubt  that  the  practice  of 
using  human  excrement  for  manuring  vegetable  gardens  entails 
a  danger  no  less  real  because  often  unrecognized." 

Necessity  of  Sewage  Disposal.— Sewage  is  obnoxious  to  the  senses 
because  of  its  appearance,  and  especially  because  on  decomposing 
it  yields  malodorous  compounds.  It  is  usually  considered  that 
hydrogen  sulphid  is  the  main  offender,  but  indol,  skatol,  cadaverin, 
mercaptan  and  some  other  compounds  are  considerably  more 
repulsive  and  exist  in  sewage. 

More  important  still  is  the  fact  that  sewage  contains  bacteria 
which  have  come  from  persons  sick  with  typhoid  fever,  dysentery, 
tuberculosis  and  other  diseases,  as  these  may  reach  the  food  or 
water  of  healthy  individuals  and  thus  give  rise  to  epidemics.  Statis- 
tics show  that  the  abandonment  of  privies  and  the  substitution 
of  a  good  sewerage  system  have  greatly  reduced  the  general  death- 
rate  in  many  a  city. 

What  Should  be  Accomplished  in  Sewage  Disposal— The  sanitary 
engineer  attempts  to  dispose  of  sewage  as  rapidly  as  possible,  with 
the  least  nuisance  to  the  smallest  number  of  people,  with  the  least 
damage  to  health  or  property,  and  at  the  smallest  cost.  Sewage 
can  be  made  entirely  harmless  only  by  the  complete  destruction 
of  its  organic  matter  and  bacteria.  A  complete  purification  is 
not  attempted  normally  as  the  plant  required  for  such  would  be 
so  elaborate  and  too  expensive.  Moreover,  practical  experience 
has  shown  this  to  be  unnecessary. 

Methods  of  Disposal.— The  method  selected  for  sewage  disposal 
will  vary  with  the  district,  location  and  means  at  the  disposal  of 
the  sanitary  engineer.  However,  in  all  cases  he  must  keep  in 
mind  convenience  and  public  health.  In  rural  districts  the  well- 
constructed  cesspool  may  of  necessity  be  used,  whereas  in  the 
urban  district  this  may  not  be  tolerated.  One  of  the  readiest 
methods,  and  the  one  which  until  the  last  few  years  has  been  uni- 
versally used  in  this  country,  is  to  allow  the  sewage  to  flow  without 
treatment  into  the  nearest  stream,  lake  or  harbor.  This  is  very 
successful  as  long  as  the  quantity  is  not  excessive,  the  dilution 
great,  and  the  receiving  water  is  not  used  by  other  communities 
for  drinking  and  culinary  purposes.  Where  this  method  is  used 
the  dilution  should  be  great.  The  Chicago  drainage  canal  was 
designed  on  the  basis  of  3.3  cubic  feet  per  second  for  1000  people. 
The  efficiency  of  purification,  however,  varies  with  the  nature  of 
the  sewage.  The  presence  of  trade  wastes,  especially  those  of  an 
oily  nature,  which  float  on  the  surface,  may  form  scums  which 
interfere  with  the  absorption  of  oxygen  from  the  air.  Rapidly 


METHODS  OF  DISPOSAL  367 

flowing  streams,  on  account  of  their  absorption  of  oxygen,  tend  to 
purify  themselves  more  rapidly  than  do  slower  ones.  Cold  water 
holds  more  oxygen  than  does  warm,  and  fresh  than  salt  water; 
hence,  there  is  a  greater  tendency  for  oxidation  in  cold  fresh  waters 
than  in  warm  or  salty  waters. 

There  is,  however,  a  growing  demand  that  sewage  be  treated 
before  it  is  thrown  into  streams  or  lakes.  This  may  be  done  by 
various  methods,  such  as  sedimentation,  sub-surface  irrigation, 
broad  irrigation  and  other  means.  For  a  description  of  each 
together  with  its  relative  value  the  student  is  referred  to  any 
of  the  standard  works  on  sewage. 

REFERENCES. 

Whipple,  George  C.:     Sewage  Disposal. 
Rosenau:     Preventative  Medicine  and  Hygiene. 
Phelps,  Earle  B. :     Microbiology  of  Sewage. 
Marshall:     Microbiology. 
Folwell,  A.  Prescott:     Sewerage. 
Sam  tee,  E.  M.:     Farm  Sewage. 
Whipple,  George  C.:     Typhoid  Fever. 
Fuller,  George  W. :     Sewage  Disposal. 


CHAPTER  XXXI. 
MILK  BACTERIOLOGY. 

ABOUT  ten  billion  gallons  of  milk  are  produced  annually  in  the 
United  States,  one-fourth  of  which  is  consumed  as  milk  and  the 
other  three-fourths  as  butter  and  cheese.  The  quantity  of  milk 
consumed  varies  in  different  localities,  being  greater  in  the  North 
than  in  the  South  and  greater  in  the  country  districts  than  in  the 
city.  It  also  varies  with  different  classes,  as  seen  from  a  survey  made 
by  Williams  of  fifteen  sections  of  Rochester,  New  York.  He  found 
that  the  average  consumption  of  milk  by  21,600  individuals  was 
little  more  than  0.24  pint  per  capita.  Furthermore,  he  found  that 
the  poor  not  only  used  less  milk  and  bought  it  in  smaller  quantities 
than  the  well-to-do,  but  the  use  of  store  milk  and  of  condensed  milk 
was  largely  confined  to  the  laboring  classes.  In  other  words,  the 
people  who  most  needed  to  be  careful  in  their  buying  used  smaller 
quantities  of  the  cheapest  food  which  they  bought  in  the  most 
expensive  manner.  It  is  usually  stated  that  about  16  per  cent,  of 
the  average  dietary  in  the  United  States  consists  of  milk  and  milk 
products,  yet  the  average  daily  consumption  per  capita  of  milk  as 
such  is  only  0.6  pint,  which  is  about  half  what  it  should  be. 

Milk  as  Food.— Milk  has  been  regarded  from  the  earliest  times  as  a 
most  important  article  of  food,  and  although  little  was  known  as  to 
its  chemical  composition  previous  to  the  eighteenth  century,  the 
ancients  attributed  many  and  peculiar  hidden  virtues  to  it. 

Good  whole  milk  or  skimmed  milk  are  among  the  best  and  cheap- 
est of  foods.  Good  fresh  milk  is  all  but  essential  to  the  welfare  of 
young  children,  and  to  the  babe  that  for  any  reason  is  deprived 
of  its  mother's  milk,  cows'  milk  is  practically  indispensable.  The 
reason  is  due  to  its  composition.  The  composition  of  human  and 
cow's  milk  is  as  follows : 

Fat,  Lactose,  Protein,  Ash, 

per  cent.        per  cent.  per  cent.  per  cent. 

Human  milk     ....     2-4  6.0-7.5  0.7-1.5  0.15-0.30 

Cows' milk       ....      3-6  3.5-5.0  2.5-4.0  0.66-0.77 

Besides  these  substances  both  cows'  and  mothers'  milk  carry 
organic  substances  which  contain  little  or  no  nitrogen,  one  of  which 
is  soluble  in  ether  and  alcohol,  the  other  in  water.  The  true  chemical 
nature  of  these  is  unknown.  The  amount  of  these  substances  in 
human  milk  at  the  beginning  of  lactation  is  about  1  per  cent.;  in 


MILK  AS  FOOD 


369 


the  middle  period  of  lactation  about  0.5  per  cent.    Cows'  milk  at 
the  middle  of  lactation  contains  about  0.3  per  cent. 

It  is  usually  stated  that  one  quart  of  milk  is  about  equal  in  food 
value  to  any  one  of  the  following: 


Salt  codfish       .      . 

Fresh  fish    .      .      . 

Chicken       .      .      . 

Beets     .      .      .      . 

Turnips. 

Butter          .      .      . 

Wheat  flour      .      . 

Cheese  .... 

Lean  round  beef    . 

Potatoes 

Spinach 

Lettuce 

Cabbage 

Eggs       .      .      .      . 


2  pounds 

3  pounds 
2  pounds 

4  pounds 

5  pounds 
£  pound 
i  pound 
^  pound 

1  pound 

2  pounds 

6  pounds 

7  pounds 
4  pounds 

8  pounds 


TOTAL  „ 
SOLIDS 


WATER 


FIG.  44. — Composition  of  cow's  milk,  showing  variations.  (Report  on  milk 
investigation,  Boston  Chamber  of  Commerce,  1915.)  (MacNutt,  The  Modern 
Milk  Problem.) 

This,  however,  considers  milk  only  from  the  total  calories  yielded. 

Digestibility  and  assimilation  must  be  considered  as  well  as  chemical 

composition  and  caloric  value;  when  this  is  done  milk  ranks  even 

higher  than  suggestec}  by  the  foregoing.    Moreover,  milk  has  other 

24 


370  MILK  BACTERIOLOGY 

functions  and  furnishes  essential  constituents  to  the  growing  animal 
which  is  not  furnished  by  many  other  foods  and  which  cannot  be 
measured  in  heat  units.  It  contains  "vitamins"  or  "accessories," 
substances  belonging  to  a  group  of  agents  which  are  widely  dis- 
tributed in  nature  and  which  are  now  regarded  as  essential  factors 
in  diet. 

Hopkins  clearly  demonstrated  that  the  feeding  of  very  small 
quantities  of  milk  to  rats  which  had  been  living  on  a  diet  inadequate 
for  normal  growth  brought  about  a  rapid  growth  in  the  animals. 
Moreover,  Osborne  and  Mendel  in  their  extensive  experiments  on 
the  growth  of  animals  have  for  several  years  been  employing 
"protein-free  milk"  as  an  indispensable  ingredient  of  their  basic 
diet  to  which  certain  isolated  food  substances  are  added.  They  find 
that  no  artificial  imitation  of  this  natural  mixture  has  been  devised 
to  replace  it  satisfactorily  for  considerable  periods  of  time.  The 
weight  and  health  of  adult  rats  can  be  maintained  for  many  months 
on  a  ration  consisting  of  protein,  starch,  sugar,  protein-free  milk, 
and  lard.  Young  animals  kept  on  this  mixture  decline  after  a  time. 
If,  however,  butter  is  substituted  for  the  lard  growth  is  resumed. 
The  active  constituent  is  the  fat  soluble  vitamin  of  the  butter  in 
contrast  to  the  water  soluble  accessories  present  in  the  protein-free 
milk.  Ordinary  skimmed  milk  contains  both  the  fat-soluble  and 
water-soluble  accessories.  The  influence  of  milk  and  sour  milk  upon 
the  growth  of  chicks  is  seen  from  the  following  summary  of  a 
great  many  tests  made  by  Rettger: 

Gain  per  chick,  Pounds. 

Fed  sour  milk 0.48 

Fed  sweet  milk 0.44 

Given  no  milk 0 . 39 

Moreover,  individuals  who  have  lived  to  extreme  old  age  have 
used  milk  in  some  of  its  forms.  Several  French  laborers  whose  diet 
consisted  largely  of  milk  lived  to  be  one  hundred  and  ten  years  or 
over.  There  are  also  authentic  records  of  a  number  of  individuals 
in  the  Balkans,  Persia,  Arabia,  and  in  the  Caucasus  Mountains  who 
have  reached  extreme  old  age,  whose  main  diet  was  milk. 

Scientists  have  long  studied  the  habits  of  these  centenarians  and 
their  diet  was  found  to  contain  large  quantities  of  sour  milk.  Metch- 
nikoff  attributes  their  long  life  as  due  to  specific  bacteria  taken  into 
the  alimentary  tract  with  the  sour  milk,  and  the  organism,  Bacillus 
bulgaricus,  is  sometimes  known  as  "the  bacillus  of  long  life"  and  is 
often  used  by  the  physician  in  combating  certain  digestive  disturb- 
ances—sometimes with  good  effects  and  at  other  times  without. 
The  cause  of  these  failures  is  only  at  the  present  time  being  fully 
understood. 

Even  the  acid-forming  bacteria  cannot  gain  the  ascendency  when 
growing  on  a  protein-rich  diet,  but  if  grown  on  a  carbohydrate  diet 


CLASSES  OF  MILK  371 

soon  produce  sufficient  acid  to  check,  if  not  kill,  the  putrefiers  which 
give  rise  to  ptomaines. 

Milk  undoubtedly  owes  its  beneficial  action  to  its  lactose  which 
is  slowly  absorbed  and  hence  regulates  the  biochemical  changes 
which  take  place  in  the  lumen  of  the  intestines.  Hull  and  Rettger 
have  conclusively  demonstrated  that  a  high  lactose  diet  markedly 
influences  the  intestinal  flora  of  man. 

Hence,  nothing  should  be  done  or  said  to  decrease  the  consump- 
tion of  milk,  but  much  should  be  done  to  see  that  the  milk  consumed 
is  pure,  clean,  and  free  from  disease-producing  bacteria.  For  although 
milk  is  one  of  the  cheapest  and  best  of  foods  it  is  responsible  for 
more  sickness  and  deaths  than  perhaps  all  other  foods  combined. 

Classes  of  Milk.— Milk  is  often  roughly  divided  into  three  classes, 
depending  upon  the  care  exercised  in  its  production  and  handling- 
certified  milk,  inspected,  or  guaranteed  milk,  and  common  milk. 

Certified  milk  has  no  unusual  properties  other  than  those  of 
exceptional  cleanliness  and  purity.  It  is  milk  which  has  been  pro- 
duced according  to  the  regulations  and  under  the  supervision  of  a 
medical  milk  commission.  The  cows  from  which  the  milk  is  pro- 
duced are  tuberculin-tested.  The  stable  and  cows  are  kept  extremely 
clean  and  no  dust  is  allowed  in  the  stable  at  the  time  of  milking. 
Small-top  sterilized  pails  are  used.  The  cows  are  carefully  groomed 
long  enough  before  milking  to  let  the  dust  settle.  The  cow's  udder 
and  flanks  are  washed  just  before  the  milking.  The  milker  wears  a 
white  suit  and  washes  his  hands  before  milking  each  cow.  The  milk 
is  cooled  either  before  or  after  bottling.  The  caps  are  so  constructed 
that  they  completely  cover  the  top  of  the  bottle,  and  many  dairies 
use  a  double  cap.  The  caps  are  sterilized  before  use  and  the  milk 
is  kept  cool  during  transit.  The  number  of  bacteria  should  not 
exceed  10,000  per  c.c.  of  milk.  Moak  gives  the  average  count  of 
321  samples  of  certified  milk  delivered  in  Brooklyn  during  1910  as 
4095  bacteria  per  c.c. 

Such  milk  is  as  near  pure  as  it  is  possible  to  produce  it  on  a  com- 
mercial scale,  and  although  it  is  required  that  it  be  delivered  to  the 
consumer  within  thirty  hours  after  production,  yet  it  will  keep  for  a 
great  length  of  time.  At  the  Paris  Exposition  in  1900  certified  milk 
from  the  United  States,  to  the  astonishment  of  the  judges,  was 
placed  on  exhibition  in  perfectly  sweet  condition  after  a  journey  of 
fourteen  to  eighteen  days,  or  3000  to  4000  miles,  in  midsummer. 

It  is  probable  that  in  none  of  our  large  cities  does  the  production 
of  certified  milk  exceed  1  per  cent,  of  the  total  supply.  This  is  due 
to  the  greater  price  which  must  be  charged  for  such  milk,  and  the 
tendency  at  the  present  tim^s  to  produce  a  high  grade  of  milk 
under  less  ideal  condition  sm  Ik  can  be  sold  at  a  more  moderate 


price. 
This  is  being  met  in  the^Wted,  inspected  or  guaranteed  milk 


372  MILK  BACTERIOLOGY 

which  is  being  placed  on  the  market.  This  is  milk  produced  from 
herds  free  from  tuberculosis  and  which  are  housed  and  cared  for 
under  good  sanitary  conditions.  Nearly  as  great  care  is  taken  in 
its  production  as  in  that  of  certified  milk.  Some  milk  so  produced 
compares  favorably  with  certified  milk. 

Common  milk  is  all  milk  not  classified  under  the  preceding 
heads  and  may  vary  in  microbial  content  from  a  few  thousand  to 
many  millions.  The  number  and  kind  vary  with  the  different 
dairies  which  produce  the  milk  and  often  with  the  city  or  state  in 
which  it  is  produced,  depending  upon  the  nature  of  the  law  and  the 
strictness  with  which  it  is  enforced. 

The  number  of  bacteria  reported  by  Hill  and  Slack  for  Boston 
milk  is  given  below : 

Per  cent. 

Below  100,000  bacteria  per  c.c 42.00 

Between  100,000  and  500,000  per  c.c 29.75 

Between  500,000  and  1,000,000  per  c.c 9.75 

Between  1,000,000  and  5,000,000  per  c.c 12.75 

Above  5,000,000  per  c.c .      .  5.00 

Uncountable  plates     .     • 0.75 

Bacteria  in  Milk. —Milk  is  one  of  the  best  foods  for  man.  It  is  also  an 
excellent  food  for  bacteria,  as  is  seen  from  the  facts  that  millions  are 
often  found  in  a  few  drops,  and  in  many  cases  the  bacteriologist  finds 
it  one  of  the  best  mediums  on  which  to  grow  his  laboratory  cultures. 
Therefore,  milk  should  be  protected  from  substances  which  contain 
bacteria,  especially  the  disease-producing  ones.  It  is  the  methods 
by  which  they  enter  and  the  speed  with  which  they  multiply  that 
we  want  to  consider.  But  it  should  be  stated  at  the  outset  that  large 
numbers  of  bacteria  in  milk  indicate  dirt,  lack  of  refrigeration,  or 
age.  It  may  or  may  not  contain  the  germs  of  disease,  but  there  is  the 
possibility.  So  milk  with  a  high  bacterial  content  is  not  necessarily 
harmful,  but  when  used  as  a  food— particularly  for  children— is  a 
hazard  too  great  to  be  countenanced,  or,  as  stated  by  Conn :  "  Good, 
clean,  fresh  milk  will  have  a  low  bacterial  count,  and  a  high  bacterial 
count  means  dirt,  age,  disease,  or  temperature.  A  high  bacterial 
count  is,  therefore,  a  danger  signal  and  justifies  the  health  officer  in 
putting  a  source  with  a  persistently  high  bacterial  count  among  the 
class  of  unwholesome  milk." 

The  number  of  bacteria  occurring  in  milk  varies  with  age,  initial 
contamination,  the  care  with  which  it  is  handled  and  kept,  tempera- 
ture, and  age.  Milk  may  contain  only  a  few  or  millions  in  each  drop, 
or  some  market  milks  at  times  contain  as  many,  but  not  as  danger- 
ous, organisms  as  sewage. 

Initial  Contamination.— The  sourq^qf  bacteria  in  milk  are:  (1) 
Intramammary,  (2)  introduced  during  milking  process,  (3)  from 
milk  utensils,  (4)  from. the  use  of  special  milk  apparatus,  (5)  con- 


INITIAL  CONTAMINATION  373 

lamination  in  transit,  (6)  contamination  on  sellers'  or  consumers' 
premises. 

Milk  as  it  is  secreted  is  a  sterile  fluid,  but  it  is  fairly  well  estab- 
lished that  as  it  is  excreted  from  the  udder  it  is  not  sterile.  Harding 
and  Wilson  examined  1230  samples  from  the  udders  of  78  cows 
which  showed  an  average  of  428  bacteria  per  c.c.  The  numbers  vary 
widely  with  different  cows,  some  yielding  milk  with  as  few  as.  25  per 
c.c.,  whereas  others  yielded  milk  with  bacterial  contents  up  to  100,- 
000.  The  organisms  obtained  from  the  healthy  udder  are  non- 
pathogenic  and  are  almost  invariably  staphylococci,  streptococci, 
and  other  forms  of  cocci.  It  is  regarded  as  certain  that  the  origin 
of  these  bacteria  is  from  the  outside  of  the  teat.  They  find  their 
way  in  through  the  orifice  of  the  teat  and  extend  up  the  milk  column, 
thus  infecting  the  milk  cistern  and  ultimately  the  ramifications  of  the 
milk  tubes  through  the  udder.  The  work  of  Savage  makes  it  appear 
that  the  number  found  in  freshly  drawn  milk  is  determined  by  the 
numbers  entering  the  teat,  and  the  selective  action  of  the  specific 
animal. 

The  bacteria  introduced  during  the  milking  process  are  derived 
from  (a)  the  coat,  udder,  and  teats  of  the  cows,  (6)  from  the  milking 
shed  and  clothes  of  the  milker,  and  (c)  from  the  hands  of  the  milker. 
It  is  impossible  to  produce  clean  milk  from  cows,  the  color  of  which 
cannot  be  distinguished  even  a  few  rods  away  because  of  the  filthy 
condition  of  their  coat.  Even  where  the  animal  is  in  a  fairly  clean 
condition  the  wiping  of  the  udder  just  before  milking  greatly  reduces 
the  number  of  bacteria  in  the  milk.  An  average  of  thirteen  experi- 
ments at  the  Storrs  Experiment  Station  yielded  the  following 
results : 

Bacteria  in  milk 
per  c.c. 

Unwiped  udders •  7058 

Wiped  udders .716 

Decrease  due  to  wiping 6342 

Numerous  investigators  have  shown  the  presence  of  bacteria  in 
large  numbers  in  cowsheds,  and  many  individuals  have  seen  stables 
or  milk  houses  in  which  each  beam  of  light  passing  through  the 
crevices  seems  to  be  filled  with  myriads  of  dancing  specks.  These 
dust  particles  carry  bacteria  and  will  increase  the  bacteria  content 
of  milk.  However,  recent  work  at  the  New  York  and  Illinois 
Experiment  Stations  has  demonstrated  that  under  fair  conditions 
this  is  a  negligible  factor. 

Then  the  hands  of  the  milker  may  not  be  quite  clean,  or  perchance 
they  have  come  in  contact  with  disease  germs  from  his  own  or  some 
one's  else  body,  and  these  may  find  their  way  into  the  milk  and  at 
times  multiply  with  an  enormous  rapidity. 

The  influence  of  the  milker  in  adding  bacteria  is  clearly  illustrated 


374  MILK  BACTERIOLOGY 

by.  the  following  experiment  reported  by  Stocking.  The  average  of 
19  tests  with  two  milkers,  one  who  had  had  no  training  in  dairy  sanita- 
tion, and  one  who  had,  showed  17,105  bacteria  per  c.c.  for  the 
untrained  man  and  2455  for  the  trained  man.  The  only  difference 
was  the  knowledge  possessed  by  the  trained  man. 

Even  more  important  than  the  surroundings  in  contaminating 
milk  are  the  utensils.  Many  buckets  are  wrongly  constructed  or 
not  scalded  each  time  so  that  every  seam  contains  hidden  away 
millions  of  bacteria.  These  immediately  grow  on  reaching  the 
fresh,  warm  milk.  Then  the  strainer  may  contain  a  good  seeding 
of  bacteria.  It  would  be  a  great  step  in  advance  could  the  strainer 
by  some  means  be  done  away  with,  for  then  greater  care  would  be 
taken  in  the  production  of  milk;  otherwise,  it  would  be  unsalable. 
The  condition  is  somewhat  similar  to  that  which  existed  when  it  wus 
first  suggested  that  bread  be  wrapped.  There  was  a  baker's  con- 
vention and  the  subject  had  come  up  for  consideration  and  the 
members  had  practically  agreed  that  all  bread  offered  by  them  should 
be  wrapped,  when  an  old  veteran  arose  and  said,  "If  we  wrap  our 
bread  in  white  paper  and  handle  it  as  we  do  now  the  paper  will  be 
so  dirty  that  when  it  reaches  the  consumer  he  will  refuse  to  buy." 
So  it  is  with  milk;  if  it  had  to  be  sold  in  the  condition  in  which  it 
comes  at  times  from  the  barn,  it  would  be  refused.  Not  that  the 
strainer  reduces  the  number  of  bacteria  in  the  milk,  for  it  does  not. 
It  only  removes  the  particles  which  are  visible  to  the  naked  eye  after 
they  have  been  washed  nearly  free  from  bacteria. 

Prucha  and  coworkers  studied  the  influence  of  all  the  utensils 
that  normally  come  into  contact  with  the  milk  both  at  the  barn 
and  at  the  dairy.  They  found  that  when  they  were  all  carefully 
steamed  the  germ  content  of  the  milk  in  the  bottles  was  about  4566 
bacteria  per  c.c.  When  similar  conditions  obtained,  except  that  the 
steaming  of  the  utensils  was  omitted,  the  germ  content  of  the  milk 
approximated  257,240  bacteria  per  c.c. 

Of  all  the  various  utensils  coming  into  contact  with  the  milk  at 
the  barn  and  at  the  dairy,  it  was  found  that  the  clarifier  and  the 
bottle-filler,  when  unsteamed,  proved  to  be  the  most  prolific  sources 
of  contamination. 

It  would,  therefore,  seem  that  the  most  important  factor  in 
producing  good  milk  is  the  scrupulous  cleanliness  of  the  milk  uten- 
sils and  not  so  much  surroundings,  as  has  been  so  much  taught  in  the 
past. 

It  is  difficult  to  accurately  measure  the  contamination  in  transit 
and  on  the  sellers'  premises,  but  it  is  quite  evident  that  at  times  it  is 
large.  Orr  reported  average  increases  as  high  as  22.7  per  cent., 
whereas  it  should  be  zero  under  ideal  conditions. 

Growth  of  Bacteria  in  Milk.— Saprophy tic  and  many  pathogenic 
bacteria  multiply  in  milk  so  that  the  number  found  in  milk  is 


CHANGES  PRODUCED  IN  MILK  &Y  BACTERIA          375 

governed,  in  addition  to  the  factors  considered  above,  by  age  and 
temperature.  The  influence  of  temperature  is  illustrated  by  the 
following: 

Temperature 

maintained  for  Bacteria  per  c.c.  at     Hours  to  curdle 

twelve  hours  F.  end  of  twelve  hours.          at  70°  F. 

40 .  4,000  75 

45 9,000  75 

50 18,000  72 

55 38,000  49 

60 453,000  43 

70 8,800,000  32 

80 55,300,000  28 

All  of  these  samples  at  first  contained  the  same  number  of  bacteria 
but  were  kept  for  twelve  hours  at  the  different  temperatures  and 
then  all  maintained  at  the  high  temperature.  We  find  over  ten 
thousand  times  as  many  bacteria  at  the  end  of  twelve  hours  in  the 
sample  kept  at  a  high  temperature  as  the  one  kept  at  a  low. 
Although  the  difference  in  temperature  was  maintained  for  only 
twelve  hours,  the  milk  at  40°  kept  three  times  as  long  as  did  that  at 
80°. 

Changes  Produced  in  Milk  by  Bacteria.— The  changes  occurring 
in  milk  are  governed  by  the  specific  bacterial  flora  which  it  contains 
and  the  temperature  at  which  it  is  kept.  Normal  clean  milk,  if 
kept  at  a  temperature  of  between  10°  and  21°  C.,  passes  through  a 
sequence  of  changes  which  can  be  divided  into  four  stages. 

First  Stage.— The  first  of  these  is  known  as  the  germicidal  stage, 
and  lasts  a  few  hours  after  the  milk  has  been  drawn  from  the  udder. 
During  this  stage  there  is  a  decrease  in  the  number  of  organisms, 
as  shown  by  the  plate  method.  The  extent  of  this  decrease  varies 
with  the  milk  of  different  cows  and  the  temperature  at  which  the 
milk  is  kept.  The  higher  the  temperature,  the  more  marked  the 
decrease,  the  sooner  the  end  of  the  germicidal  period  is  reached. 
There  is  a  great  difference  in  opinion  among  bacteriologists  con- 
cerning the  nature  of  the  phenomenon.  Some  would  account  for 
it  on  the  grounds  that  milk  is  a  favorable  cultural  media  for  many 
bacteria,  but  not  all.  The  ones  for  which  it  is  unsuited  rapidly 
die  off.  Others  consider  that  the  milk,  like  the  blood  and  many 
other  body  fluids,  possesses  bactericidal  power  which  is  very  weak 
and  soon  lost.  Rosenau  and  McCoy,  however,  consider  that  the 
bacteria  are  agglutinated  and  not  killed.  On  plating,  the  clump 
gives  rise  to  the  colony  in  place  of  each  individual  organism,  as  is  nor- 
mally the  case. 

This  germicidal  power  is  lost  on  boiling  the  milk  or  heating  to  a 
temperature  of  80°  C.,  and  some  have  urged  this  as  an  objection 
against  pasteurization,  but  in  the  "holder"  process  this  is  not  a 
warranted  objection. 


376  MILK  BACTERIOLOGY 

Second  Stage.— This  stage  extends  from  the  end  of  the  germicidal 
period  to  the  time  of  curdling.  There  may  be  a  gradual  increase 
during  this  time  of  many  species,  but  the  predominating  types  are 
the  Bad.  lactis  acidi.  These  rapidly  produce  lactic  acid  which 
exerts  a  suppressing  influence  on  many  species.  When  the  milk 
reaches  an  acidity  of  .75  to  .80  per  cent,  it  usually  curdles.  The  lactic 
acid  organisms  seldom  produce  more  than  1.25  per  cent.  acid. 

Third  Stage.— This  stage  extends  from  the  time  of  curdling  until 
the  neutralization  of  the  acid.  The  acidity  becomes  so  great  that 
the  action  of  the  lactic  acid  bacteria  is  checked  and  their  number, 
which  at  first  may  be  as  high  as  100,000,000  per  c.c.,  rapidly 
decreases.  The  predominating  species  become  O'idium  lactis,  certain 
species  of  molds  and  yeasts.  The  proteins  are  broken  down  with 
the  formation  of  ammonia  which  neutralizes  the  acidity. 

Fourth  Stage.— The  liquefying  and  peptonizing  bacteria  which 
remained  inactive  in  the  sour  milk  find  suitable  conditions  in  the 
alkaline  media  for  their  growth.  They  rapidly  decompose  the 
casein. 

Abnormal  Changes  in  Milk.— At  times  foreign  undesirable  organ- 
isms find  their  way  into  milk  and  produce  abnormal  and  objectionable 
changes.  The  B.  coli  communis  and  the  Bad.  lactic  aerogenes  types 
produce  considerable  gas  and  disagreeable  odors  and  flavors  in  the 
milk.  B.  lactis  viscosus  produces  a  slimy  or  ropy  condition  of  the 
milk.  The  slimy  condition  is  supposed  to  be  due  to  the  mucin  con- 
taining capsule  which  surrounds  these  bacteria.  Milk  may  be  normal 
in  color  when  first  produced,  but  on  standing  may  turn  blue  due  to 
B.  cyanogenes  or  red  due  to  B.  erythrogenus  or  B.  prodigiosus.  At 
times  a  bitter  taste  develops  in  milk  some  time  after  it  has  been 
drawn  from  the  udder.  This,  according  to  Conn,  is  due  to  a  micro- 
coccus. 

Although  these  changes  are  very  objectionable  when  considered 
from  the  standpoint  of  the  dairymen,  they  are  not  known  to  be  the 
cause  of  illness.  However,  when  milk  putrefies  with  the  production 
of  a  bitter  alkaline  milk  illness  often  does  result  from  its  use.  This 
may  be  due  to  the  poisonous  action  of  the  ptomaines  which  it  con- 
tains, or  probably  more  often  to  the  bacterial  infection. 

Classes  of  Bacteria.— The  bacteria  found  in  milk  are  a  hetero- 
geneous lot  but,  according  to  Hastings,  may  be  roughly  divided 
into  five  classes,  as  follows: 

1.  Acid-forming  Bacteria.— There  are  constantly  present  in  milk 
many  acid-forming  bacteria.  These  vary  in  morphology,  cultural 
characteristics,  and  products  of  fermentation.  They  may  be  divided 
into  five  groups.  The  number  and  kind  vary  greatly  in  milk, 
depending  upon  the  methods  of  handling. 

(a)  The  most  important  organism  of  this  group  is  Bad.  lactis 
acidi.  The  group,  however,  includes  a  number  of  organisms.  They 
produce  no  gas,  a  mild  acid  flavor,  and  are  desirable. 


ABNORMAL  CHANGES  IN  MILK  377 

(b)  The  best  known  representatives  of  this  group  are  B.  coli 
communis  and  Bad.  lactis  aerogenes.    These  organisms  give  to  milk 
a  sharp  tang  and  are  the  particular  enemies  of  the  cheese  maker  as 
they  are  the  cause  of  gassy  curd.    They  are  especially  numerous  in 
milk  which  is  produced  and  handled  under  unsanitary  conditions 
and  in  such  milk  outnumber  those  of  Group  a,  but  the  rapid  growth 
and  acid  production  of  Group  a  soon  checks  them. 

(c)  This  is  represented  by  Bacillus  bulgaricus  and  the  rod-shaped 
organisms  which  have  been  especially  studied  by  de  Freudenreich. 
They  produce  a  curd  which  is  easily  broken  by  shaking  and  shows  no 
tendency  to  express  whey.    They  give  to  the  milk  a  pleasant  acid 
flavor  and  are  desirable. 

(d)  Acid-forming   Udder  Bacteria.— These  are  the  characteristic 
bacterial  flora  of  the  healthy  udder  and  consist  mainly  of  cocci  with 
few  bacilli.    They  are  slow  growers  and  may  curdle  milk,  but  the 
curd  so  formed  resembles  that  formed  by  rennet.    They  produce 
acetic,  propionic,  butyric,  and  caproic  acid  but  no  lactic  acid. 
They  are  an  unimportant  group  of  organisms,  so  far  as  the"  milk 
is  concerned. 

2.  Peptonizing    Bacteria.— These    organisms    digest    the    casein 
either  with  or  without  coagulation  at  times  with  the  formation  of 
an  alkaline  reaction.    Most  of  these  are  bacilli  of  various  shapes 
and  sizes,  some  of  them  being  the  largest  organism  found  in  milk. 
There  are  both  motile  and  non-motile  varieties.     Many  develop 
very  strong  putrefactive  odors.    Barny  or  cowy  odors  are  caused  by 
this  type  of  bacteria.    They  are  all  undesirable  and  their  presence 
in  milk  indicates  unsanitary  condition  of  production  and  handling. 

3.  Bacteria  Producing  Milk  of  Unusual  Character.— Occasionally 
bacteria  which  produce  abnormal  changes  or  so-called  "  diseases' ' 
of  milk  find  their  way  into  milk  from  unclean  surroundings.    They 
produce  various  queer  milks,  for  example,  red,  blue,  and  green. 
Sometimes  milk  develops  a  bitterness  after  it  is  drawn.    This  is  due 
to  the  products  from  a  number  of  bacteria  and  yeast.    At  other  times 
milk  is  changed  to  a  slimy  or  ropy  consistency  and  may  at  times 
result  in  considerable  economic  losses.    These  organisms  are  quite 
resistant  to  heat  and  frequently  pass  uninjured  through  the  ordinary 
methods  of  cleaning  and  scaldings.    Because  of  this,  dirty  utensils 
once  infected  become  a  constant  source  of  infection. 

4.  Inert  Organisms. —These  are  mostly  cocci  which  produce  no 
appreciable  change  in  milk  and  are  unimportant. 

5.  Pathogenic  Bacteria.— This  class  consists  of  the  pathogenic 
bacteria,  B.  dysenteric  shiga,  B.  dysenteric  flexner,  B.  typhosus,  B. 
paratyphosus  a.  and  B.  paratyphosus  £.,  V.  cholerc,  Bact.  diphtheria, 
Bad.  tuberculosis,  B.    lactimorbimic  melitensis.     These  organisms 
produce  no  perceptible  change  in  the  milk  in  which  they  grow  but 
are  dangerous  and  may  give  rise  to  epidemics. 


CHAPTER   XXXII. 
MILK  AND  DISEASE. 

ALTHOUGH  milk  is  one  of  the  cheapest  and  best  of  foods,  yet  it  is 
responsible  for  more  sickness  and  death  than  perhaps  all  other  foods 
combined.  The  reasons  for  this  have  been  summarized  by  Rosenau 
as  follows : 

"1.  Bacteria  grow  well  in  milk;  therefore,  a  very  slight  infection 
may  produce  widespread  and  serious  results.  (2)  Of  all  foodstuffs, 
milk  is  the  most  difficult  to  obtain,  handle,  transport,  and  deliver 
in  a  clean,  fresh,  and  satisfactory  condition.  (3)  It  is  the  most 
readily  decomposable  of  all  our  foods.  (4)  Finally,  milk  is  the  only 
standard  article  of  diet  obtained  from  animal  sources  consumed  in 
its  raw  state." 

Diseases  conveyed  through  milk  are  of  two  classes:  (1)  Definite 
diseases  of  animal  origin— tuberculosis,  foot-and-mouth  disease, 
malta  fever,  and  anthrax,  and  indefinite  ailments  as  diarrheal  infec- 
tions and  probably  contagious  abortion.  (2)  Diseases  of  human 
origin— typhoid  fever,  paratyphoid  fever,  diphtheria,  scarlet  fever, 
tuberculosis,  septic  sore  throat,  and  possibly  others. 

Sources  of  Infection.:— Infection  of  bovine  origin  is  very  common, 
especially  in  the  case  of  tuberculosis  wherein  the  animal  is  suffering 
with  open  cases  of  this  disease  and  the  organism  gets  into  the  sur- 
roundings from  the  respiratory  or  alimentary  tract.  Extreme  care 
in  the  milking  process  may  decrease  the  infection  from  this  source, 
but  not  so  in  the  case  of  tuberculosis  of  the  udder,  which  probably 
accounts  for  the  main  cases  where  the  tubercle  bacilli  find  their  way 
into  milk. 

As  a  rule  milk  becomes  infected  from  human  sources.  This  may 
be  either  direct  or  indirect  human  infection. 

Direct  human  infection'  may  come  from  a  person  either  suffering 
with  the  disease  or  carrying  the  infective  organism.  The  more 
common  are  the  following: 

1.  The  most  common  method  is  where  the  milkers  or  other 
handlers  of  milk  are  suffering  with  a  communicable  disease  in  a  mild 
unrecognized  condition. 

2.  A  second  common  source  of  infection  is  where  the  milker  or 
vender  of  milk  has  been  brought  in  contact  with  sufferers  of  com- 
municable diseases  and  still  attends  to  his  regular  work  in  the  hand- 
ling of  milk. 


CHARACTER  OF  MILK-BORNE  DISEASE  379 

3.  A  third  and  probably  very  important  source  of  infection  comes 
from  carriers  who  work  on  the  farms,  in  dairies,  or  other  places 
where  milk  is  handled. 

Indirect  human  infection  comes  largely  from  the  use  of  infected 
water  which  is  used  in  the  washing  of  buckets,  bottles,  and  other 
milking  utensils.  Cows  often  have  access  to  polluted  water  and 
infection  from  this  source  may  find  its  way  into  the  milk  from  being 
on  the  body  of  the  animal. 

Character  of  Milk-borne  Diseases. —Milk-borne  diseases  have 
characteristics  which  greatly  assist  the  epidemiologist  in  his  work. 
The  most  important  are  the  following: 

1.  The  cases  usually  follow  the  route  of  the  milkman  and  it  is 
often  possible  to  plot  his  route  from  the  cases  of  the  specific  disease. 
There  would  thus  be  the  inhabitants  of  homes  where  the  infected 
milk  is  used  suffering  with  the  disease,  while  neighbors  who  use 
other  milk  escape.    There  may  be  many  purchasers  of  the  infected 
milk  who  may  escape,  but  when  careful  inquiry  is  made  it  is  found 
consumers  of  the  implicated  supply  furnish  a  much  higher  percentage 
of  cases  than  does  the  rest  of  the  community.    The  smallest  per- 
centage invasion  of  households  is  met  with  in  scarlet  fever  outbreaks. 
But  this  is  easily  explained  when  one  considers  the  number  of  missed 
cases  in  this  disease. 

2.  The   outbreaks   from   infected   milk   are    usually   explosive. 
Sometimes  the  majority  of  the  cases  occur  within  a  few  days  of  each 
other.    Usually  there  is  little  secondary  infection  and  the  decline  is 
rapid  on  removal  of  the  source  of  infection.    The  epidemic  at  Stam- 
ford, Connecticut,  in  1895,  is  a  good  example.     There  were  386 
cases  of  typhoid  fever  and  22  deaths  in  the  period  from  April  15  to 
May  28.    There  were  176  persons  stricken  during  the  first  week. 

Although  the  explosive  type  of  epidemic  is  usually  characteristic 
of  milk-borne  outbreaks,  yet  Parker  points  out  that  the  smoldering 
kind  may  be  very  commonly  due  to  infected  milk.  He  cites  as  an 
example  the  experience  of  Hill  of  North  Branch,  Minnesota,  where 
one  of  the  physicians  pointed  out  that  in  his  seventeen  years  of 
practice  during  the  first  twelve  there  was  no  typhoid  fever,  but  in  the 
last  five  years  native  cases  of  unknown  origin  had  been  frequent. 
Acting  on  this  information,  a  list  of  21  cases  of  typhoid  fever  that 
had  appeared  in  the  town  in  the  last  five  years  was  made,  and 
inquiry  showed  that  seventeen  of  the  patients  were  regular  custo- 
mers of  a  dairyman  who  came  to  town  five  years  before,  two  others 
were  irregular  customers,  and  two  others  may  have  used  milk  from 
his  dairy.  It  was  learned  that  the  wife  of  the  dairyman,  who  washed 
the  cans,  had  suffered  with  typhoid  fever  twenty-two  years  before 
and  gave  a  positive  Widal  reaction,  but  typhoid  bacilli  were  not 
isolated  from  her  stools.  She  was  forbidden  to  have  anything  more 
to  do  with  the  dairy  and  the  proprietor  was  told  that  if  another 


380  MILK  AND  DISEASE 

primary  case  developed  among  his  customers  his  dairy  would  be 
closed.  Rumors  of  the  affair  spread  through  the  town  and  his 
customers  left  him  and  the  family  moved  away,  after  which  there 
was  no  more  typhoid  fever. 

3.  It  frequently  happens  that  the  better  class  suffer  more  than 
do  the  poor,  as  they  can  afford  more  milk  and  use  it  more  freely. 

4.  There  is  a  special  incidence  among  milk  drinkers,  there  fre- 
quently being  an  individual  who  dislikes  milk  escaping,  whereas 
the  remainder  of  the  family  is  attacked. 

5.  Women  and  children  are  more  often  victims  in  milk-borne 
typhoid  than  are  the  adult  male  population  due  to  their  use  of  raw 
milk. 

6.  There  is  some  evidence  which  indicates  that  the  incubation 
period  is  shorter  and  the  mortality  lower  in  milk-borne  epidemics 
than  in  others.  » 

The  mild  character  of  the  disease  is  usually  attributed  to  the 
attentuation  of  the  pathogenic  properties  of  the  microorganisms 
through  their  growth  in  milk. 

7.  Milk  epidemics  of  typhoid  spread  over  a  rather  short  milk 
route,  whereas  when  milk  is  brought  from  a  considerable  distance 
there  is  not  the  likelihood  of  infection,  thus  indicating  that  typhoid 
germs  tend  to  disappear  from  milk  under  certain  conditions. 

Extent  of  Milk-borne  Disease.— The  extent  of  milk-borne  epidemics 
cannot  be  accurately  measured,  as  even  at  the  present  day  many 
cases  go  undetermined  or  perhaps  attributed  to  other  causes.  But 
the  experience  of  Boston,  Massachusetts,  which  has  a  fair  milk 
supply  indicates  the  gravity  of  the  subject. 

Year.                   Epidemics.  Cases. 

1907  Diphtheria 72 

1907  Scarlet  fever • 717 

1908  Typhoid  fever 400 

1910  Scarlet  fever 842 

1911  Septic  sore  throat 2064 

Total 4095 

This  indicates  that  scarlet  fever  and  septic  sore  throat  may  be 
conveyed  even  more  often  than  typhoid  fever. 

Tuberculosis  is  the  most  important  of  all  milk-borne  diseases, 
both  because  of  the  frequency  with  which  it  is  conveyed  and  the 
seriousness  of  the  disease.  It  may  be  either  bovine  or  human  in 
origin.  Human  infection  is  rarer  than  bovine,  but  it  is  certain  that  a 
tubercular  patient  may  infect  milk,  and  Hess  in  1918  actually  isolated 
the  jiuman  tuberculosis  bacillus  from  a  sample  of  market  milk. 

Koch  in  1901  announced  that  there  was  practically  no  danger  of 
man's  contracting  tuberculosis  from  cattle,  but  his  statement  was 
immediately  challenged  by  many  bacteriologists  who  have  since 
brought  forth  conclusive  evidence  of  the  falseness  of  Koch's  dictum. 


EXTENT  OF  MILK-BORNE  DISEASE 


381 


The  summarized  English,  German,  and  American  findings  in 
1511  cases  are  given  below: 


COMBINED  TABULATION  SHOWING   ORIGIN  OF   CASES  OF 
TUBERCULOSIS. 


Diagnosis  of  cases 
examined. 


Adults  sixteen        Children  five  to 
years  and  over.         sixteen  years. 


Children  under 
five  years. 


Human. 

Bovine. 

Human. 

Bovine. 

Human. 

Bovine. 

Pulmonary  tuberculosis      .      . 

778 

3 

14 

35 

1 

Tuberculosis  adenitis,  axillary  or 

inguinal     

3 

4 

2 

Tuberculosis  adenitis,  cervical 

36 

1 

36 

22 

15 

24 

Abdominal  tuberculosis 

16 

4 

8 

9 

10 

14 

Generalized  tuberculosis,  aliment- 

ary origin        

6 

1 

3 

4 

17 

15 

Generalized  tuberculosis     . 

29 

5 

1 

74 

7 

Generalized  tuberculosis,  including 

meninge,    alimentary   origin     . 

5 

.  .  . 

11 

81 

11 

Tubercular  meningitis  .... 

1 

3 

. 

28 

4 

Tuberculosis  of  bones  and  joints 

32 

1 

41 

3 

27 

Genito-urinary  tuberculosis     . 

22 

1 

2 

Tuberculosis  of  skin      .... 

10 

3 

4 

6 

2 

Miscellaneous  cases: 

Tuberculosis  of  tonsils    . 

1 

Tuberculosis  of  mouth  and  cer- 

vical nodes 

1 

Tuberculosis  sinus  or  abscess 

2 

Sepsis,   latent   bacilli     . 

1 

Totals      '     . 

940 

15 

131 

46 

292 

76 

The  percentage  incidence  of  bovine  infection  would,  therefore, 
be  as  follows: 

Adults  sixteen         Children  five         Children  under 
years  and  over,   to  sixteen  years.          five  years. 

2.8 
61.0 
58.0 


0.4 

2.7 
20.0 


Pulmonary  tuberculosis    .... 
Tuberculosis  adenitis,  cervical    . 
Abdominal  tuberculosis    .... 
Generalized  tuberculosis,  alimentary 

origin 14.0 

Generalized  tuberculosis  ....         0 
Generalized   tuberculosis,    including 

meningitis,  alimentary  origin  .      .         0 
Tubercular  mentingitis     ....         0 
Tuberculosis  of  bones  and  joints     .        3.3 
Tuberculosis  of  skin    .  23.0 


0 

38.0 
53.0 


57.0 
16.0 

0 
0 

6.8 
60.0 


47.0 


66.0 
4.6 
0 
0 


It  is  probable  that  the  majority  of  all  cases  of  bovine  tuberculosis 
in  man  are  due  to  infected  milk,  as  there  is  little  danger  from  meat 
since  it  is  usually  cooked  and  tuberculosis  of  the  muscles  is  very 
rare. 

Tuberculosis  is  quite  prevalent  among  cows,  varying  in  different 
places  from  a  few  to  as  high  as  50  per  cent,  Savage  gives  from 


382  MILK  AND  DISEASE 

Dallar  the  following  as  being  the  percentage  found  in  various  places: 
England,  20  per  cent.;  Denmark,  31  per  cent.;  Sweden,  42  per  cent.; 
Norway,  8.4  per  cent.;  Belgium,  60  per  cent.;  Massachusetts  (1897), 
58.9  per  cent. 

It  is  a  general  conception  that  tubercle  bacilli  occur  only  in  milk 
obtained  from  animals  suffering  with  tuberculosis  of  the  udder;  this 
is  not  strictly  true  as  is  seen  from  the  following  conclusions  by 
Mohler: 

"The  tubercle  bacillus  may  be  demonstrated  in  milk  from  tuber- 
cular cows  when  the  udders  show  no  perceptible  evidence  of  the 
disease  either  macroscopically  or  microscopically. 

"The  bacilli  of  tuberculosis  may  be  excreted  from  such  udder  in 
sufficient  numbers  to  produce  infection  in  experimental  animals 
both  by  ingestion  and  inoculation. 

"The  presence  of  the  tubercle  bacilli  in  the  milk  of  tubercular 
cows  is  not  constant,  but  varies  from  day  to  day. 

"Cows  secreting  virulent  milk  may  be  affected  with  tuberculosis 
to  a  degree  that  can  be  detected  only  by  the  tuberculin  test. 

"The  physical  examination  or  general  appearance  of  the  cow 
cannot  foretell  the  infectiveness  of  the  milk. 

"The  milk  of  all  cows  which  have  reacted  to  the  tuberculin  test 
should  be  considered  as  suspicious  and  should  be  subjected  to  sterili- 
zation before  using." 

Shroeder,  however,  concluded  that  only  40  per  cent,  of  the  cows 
which  react  to  the  tuberculin  test  actively  expel  tubercle  bacilli. 

Market  milk  often  contains  the  tubercle  bacilli,  as  may  be  seen 
from  the  following  table  compiled  by  Parker. 


~i~~r.v^  -, — ~^  Percentage 

"lace.                       Investigator.  examined.  positive.  positive. 

England  Macfayden  77  17  22.1 

Germany  Muller  1596  97  6.2 

Germany  Beatty  272  27  10.0 

Liverpool  Delepine  12  22  17.6 

1897             Liverpool      '  Hope  228  12  5.2 

1900            London  Klein  100  7  7.0 

St.  Petersburg  Scharbekow  80  4  5.0 

Kiew  Pawlowsky  51  1  2.0 

Krakow  Bujwid  60  2  3.3 

Naples  Marconi  14  7  50.0 

Berlin  Petri  64  9  14.0 

Berlin  Beik  56  17  30.3 

Schev  Ott  27  27  11.1 

Koniesburc1  .T«o<ror  1  nn  7  79 


1908 
1905 


TUBERCLE 

BACILLI  IN  MARKET  MILK. 

Place. 

Investigator. 

Samples 
examined. 

Number 
positive. 

England 

Macfayden 

77 

17 

Germany 

Muller 

1596 

97 

Germany 

Beatty 

272 

27 

Liverpool 

Delepine 

12 

22 

Liverpool 

Hope 

228 

12 

London 

Klein 

100 

7 

St.  Petersburg 

Scharbekow 

80 

4 

Kiew 

Pawlowsky 

51 

1 

Krakow 

Bujwid 

60 

2 

Naples 

Marconi 

14 

7 

Berlin 

Petri 

64 

9 

Berlin 

Beik 

56 

17 

Schev 

Ott 

27 

27 

Konigsburg 

Jaeger 

100 

7 

Leipzic 

Eber 

210 

22 

Rotterdam 

Smit 

567 

14 

Rotterdam 

Smit 

1584 

45 

Washington 

Anderson 

223 

15 

Louisville 

Field 

119 

46 

New  York 

Hess 

105 

17 

Philadelphia 

Campbell 

130 

18 

Chicago 

Tonney 

144 

15 

Rochester 

Goler 

237 

30 

10.5 

2.  7 


oeram  Smit  1584  45  2.8 

Washington  Anderson  223  15  6.7 

Louisville  Field  119  46  29  5 

New  York  Hess  105  17  16.2 

Philadelphia  Campbell  130  18  13.8 

Chicago  Tonney  144  15  10.5 

Rochester  rinlor  0*7  in  12.6 


THE  TUBERCULIN  TEST  383 

•i 

It  has  been  shown  that  man  is  susceptible  to  the  bovine  type  of 
tuberculosis  and  that  the  organism  often  is  found  in  market  milk, 
and  Rosenau  estimates  that  probably  7  per  cent,  of  the  cases  of 
tuberculosis  thus  have  their  origin.  The  significance  of  these 
figures  becomes  apparent  when  we  realize  that  160,000  individuals 
die  each  year  in  the  United  States  of  this  disease,  and  11,200  would 
get  their  infection  from  milk. 

This  is  a  needless  loss  of  human  life,  for  the  information  now 
available  is  sufficient  to  prevent  every  one  of  these  cases  if  milk  be 
obtained  only  from  cows  which  have  given  negative  tuberculin 
tests. 

The  Tuberculin  Test.— This  reaction  should  be  applied  to  all  cows 
and  is  carried  out  as  follows  : 

"  Inspections  should  be  carried  on  while  the  herd  is  stabled.  If  it 
is  necessary  to  stable  animals  under  unusual  conditions  or  among 
surroundings  that  make  them  uneasy  and  excited,  the  tuberculin 
test  should  be  postponed  until  the  cattle  have  become  accustomed 
to  the  new  conditions.  The  inspection  should  begin  with  careful 
physical  examination  of  each  animal.  This  is  essential,  because  in 
some  severe  cases  of  tuberculosis  no  reaction  follows  the  injection 
of  tuberculin  on  account  of  saturation  with  toxins,  but  experience 
has  shown  that  these  cases  can  be  discovered  by  physical  examina- 
tion. The  latter  should  include  a  careful  examination  of  the  udder 
and  of  the  superficial  lymphatic  glands  and  auscultation  of  the 
lungs. 

"Each  animal  should  be  numbered  or  described  in  such  a  way 
that  it  can  be  recognized  without  difficulty.  It  is  well  to  number 
the  stalls  with  chalk  and  transfer  these  numbers  to  the  transfer 
sheet,  so  that  the  temperature  of  each  animal  can  be  recorded  in 
its  appropriate  place  without  danger  of  confusion.  The  following 
procedure  has  been  used  extensively  and  has  given  excellent  results: 

(a)  "Take  the  temperature  of  each  animal  to  be  tested  at  least 
twice  at  intervals  of  three  hours  before  tuberculin  is  injected. 

(b)  "Inject  the  tuberculin  in  the  evening,  preferably  between 
the  hours  of  6  and  9  P.M.    The  injection  should  be  made  with  care- 
fully sterilized  hypodermic  syringes.    The  most  convenient  point 
for  injecting  is  back  of  the  left  scapula.    Prior  to  the  injection  the 
skin  should  be  washed  carefully  with  a  5  per  cent,  solution  of  car- 
bolic acid  or  other  antiseptic. 

(c)  "The  temperature  should  be  taken  nine  hours  after  the  injec- 
tion, and  temperature  measurements  repeated  at  regular  intervals 
of  two  to  three  hours  until  the  sixteenth  hour  after  the  injection. 

(d)  "When  there  is  no  elevation  of  temperature  at  this  time 
(sixteen  hours  after  injection)  the  examination  may  be  discontinued, 
but  if  the  temperature  shows  an  upward  tendency,  measurements 
must  be  continued  until  a  distinct  reaction  is  recognized  or  until 
the  temperature  begins  to  fall. 


384 


MILK  AND  DISEASE 


(e)  "If  the  reaction  is  detected  prior  to  the  sixteenth  hour  the 
measurements  should  be  continued  until  the  expiration  of  this 
period. 

(/)  "If  there  is  an  unusual  change  of  temperature  of  the  stable 
or  a  sudden  change  in  the  weather,  this  fact  should  be  recorded  on 
the  report  blank. 

(g)  "If  a  cow  is  in  a  febrile  condition  tuberculin  should  not  be 
used,  because  it  would  be  impossible  to  determine  whether,  if  a  rise 
in  temperature  occurred,  it  was  due  to  the  tuberculin  or  to  some 
transitory  illness. 

(h)  "Cows  should  not  be  tested  within  a  few  days" after  or  before 
calving,  for  experience  has  shown  that  the  results  at  this  time  may 
be  misleading. 


SECTION 

SHOWING 

BRACKET 

TOR 

TRAY 


FIG.  45. — Straus's  home  pasteurizer.     (From  Rosenau's  Preventative  Medicine.) 

(i)  "The  tuberculin  test  is  not  recommended  for  calves  under 
three  months  old. 

(j)  "In  old,  emaciated  animals  and  in  retests  use  twice  the  usual 
dose,  for  these  animals  are  less  sensitive. 

(k)  "Condemned  cattle  must  be  removed  from  the  herd  and  kept 
away  from  those  that  are  healthy. 

1.  "In  making  postmortems  the  carcasses  should  be  thoroughly 
inspected  and  all  the  organs  should  be  examined. 

"No  animal  whose  temperature  exceeds  39.5°  C.  (103°  F.)  is  a 
fit  subject  for  the  tuberculin  test. 

"A  rise  of  temperature  to  above  40°  C,  (104°  F,)  in  any  animal 


PASTEURIZATION 


385 


whose  temperature  at  the  moment  of  injection  was  below  39.5°  C. 
(103°  F.)  is  to  be  regarded  as  a  positive  reaction. 

"Any  rise  in  temperature  between  39.5°  C.  (103°  F.)  and  40°  C. 
(104°  F.)  must  be  regarded  as  of  doubtful  significance;  animals 
exhibiting  such  require  special  study." 

Milk-conveyed  typhoid  fever  can  be  handled  nearly  as  effectively 
as  can  tuberculosis  by  excluding  typhoid  carriers  as  producers  and 
handlers  of  milk.  This  can  be  very  easily  and  efficiently  done  by 
requiring  the  blood  test  for  all  dairymen  and  their  workers. 


. 
Milk 


Acid 

Coagulating  j 

Group 


Acid 
Group 


Inert 
Group 


Alkali 
Group 


Feptonizingf 
Group 


Milk  Pasteurized  fbr,30  Minutes  at 


(H5°F.)      (160°  F.) 


7(5 .7'  C. 


82.2°C. 
(180°F.) 


87.8°C. 
(190"F.) 


03.3°C> 
(200°  F.) 


FIG.  46. — The  hypothetical  relation  of  the  bacterial  groups  in  raw  and  pasteurized 
milk.     (Tanner,  after  Ayers  and  Johnson.) 


Pasteurization.— There  are  two  methods  of  pasteurization— the 

"flash"  and  "continuous"  processes.    In  the  flash  method  the  milk 

is  heated  to  80°  or  90°  C.  for  from  one  to  five  minutes  and  then 

cooled  to  50°  C.  or  below.    This  method  is  rapid,  cheap,  and  much 

25 


386  MILK  AND  DISEASE 

in  vogue  but  does  not  give  uniform  results,  is  not  entirely  reliable, 
and  does  not  meet  the  approval  of  the  sanitarian. 

The  continuous  method  is  much  to  be  preferred  and  consists  in 
heating  the  milk  to  a  temperature  of  65°  C.  for  thirty  minutes  and 
then  cooling  to  50°  C.  or  below. 

The  ideal  method  for  home  pasteurization  of  milk  is  outlined  by 
Rosenau  as  follows : 

"Pasteurization  in  the  bottle  is  the  perfection  of  the  art.  It  is 
the  ideal  method,  because  the  danger,  however  slight,  of  recontami- 
nation  is  entirely  eliminated.  In  order  to  pasteurize  milk  in  bottles 
the  bottles  must  be  well  sealed  with  a  tight  cork  and  cap,  or  equally 
effective  stopper.  The  bottles  containing  the  milk  may  either  be 
immersed  in  a  water  bath,  brought  to  the  proper  temperature,  held 
there  a  sufficient  length  of  time  and  then  chilled ;  or  the  method  used 
in  beer  pasteurization,  such  as  the  Loew  pasteurizers,  may  be  used. 
In  this  case  the  bottles  are  subjected  to  a  spray  or  shower  of  heated 
water. 

"After  the  bottles  have  been  thoroughly  cleaned  they  are  placed 
in  the  tray  (A)  and  filled  with  the  milk  or  mixture  used  for  one 
feeding.  Then  put  on  the  corks  or  patent  stoppers  without  fasten- 
ing them  tightly. 

"The  pot  (B)  is  now  placed  on  the  wooden  surface  of  the  table 
or  floor  and  filled  to  the  support  (C)  with  boiling  water.  Place  the 
tray  (A)  with  the  filled  bottles  into  the  pot  (B)  so  that  the  bottom 
of  the  tray  rests  on  the  support  (C),  and  put  cover  ( D)  on  quickly. 

"After  the  bottles  have  been  warmed  up  by  the  steam  for  five 
minutes,  remove  the  cover  quickly,  turn  the  tray  so  that  it  drops 
into  the  water,  replace  the  cover  immediately.  This  manipulation 
is  to  be  made  as  rapidly  as  possible  to  avoid  loss  of  heat.  Thus  it 
remains  for  twenty-five  minutes." 

REFERENCES. 

Rosenau :     The  Milk  Question. 

McNutt:     The  Modern  Milk  Problem. 

Savage:     Milk  and  the  Public  Health. 

Parker:     City  Milk  Supply. 

Lane,  Clayton:     Milk  and  Its  Hygienic  Relations. 

Heineman:   .Milk. 

Douglass:     The  Bacillus  of  Long  Life. 


CHAPTER  XXXIII. 
BACTERIA  IN  OTHER  FOODS. 

ALL  foods  except  those  cooked  just  previous  to  eating  contain 
bacteria,  the  number  and  kind  varying  with  the  specific  product  and 
especially  the  method  of  handling.  The  greater  part  of  such 
bacteria  are  without  special  significance.  Some  are  beneficial  and 
play  an  important  role  in  the  ripening  or  other  changes  through 
which  the  food  passes.  These  are  considered  in  a  later  chapter,  the 
present  one  being  reserved  for  a  consideration  of  those  bacteria 
which  cause  spoilage  of  food  though  not  necessarily  injurious  to  health, 
but  of  importance  from  an  economic  standpoint,  and  the  pathogenic 
organisms  which  find  their  way  into  food  and  may  infect  the  con- 
sumer. 

Bacteria  in  Butter.— Many  of  the  bacteria  which  occur  in  unclean 
milk  multiply  and  give  bad  flavors  to  the  butter  produced  from 
such  milk.  This  is  true  to  such  a  degree  that  most  dairies  first 
sterilize  their  cream  and  then  add  to  it  a  pure  culture  for  the  ripen- 
ing of  the  cream. 

Any  pathogenic  bacteria  which  find  their  way  into  the  milk  may 
persist  in  the  butter.  While  the  typhoid  organism  grows  well  in 
fresh  milk  the  increased  acid  production  tends  to  check  their  mul- 
tiplication and  may  actually  kill  many.  They  are,  however,  fairly 
resistant  to  lactic  acid,  as  seen  from  the  following  results  (Krum- 
wiede  and  Noble) : 

Reaction.  Number  of  typhoid 

Days.  Per  cent.  bacilli. 

0 1.0  392,000 

7 2.2  65,000,000 

8 5.0  300,000,000 

9 113,000,000 

10 181,000 

11 10.0  400 

Hence,  lactic  acid  cannot  be  depended  upon  to  free  butter  from 
the  typhoid  bacilli.  Moreover,  numerous  investigators  have  found 
typhoid  bacilli  in  butter  after  varying  lengths  of  time— Bolley, 
five  to  ten  days;  Heim,  twenty-one  days;  Pfulel,  twenty-four  days; 
Buck,  twenty-seven  days.  Washburn  obtained  the  organism  after 
one  hundred  and  fifty  days  from  butter  which  had  been  experimen- 
tally infected  with  typhoid  bacilli,  and  Boyd  reports  an  epidemic 
of  typhoid  fever  which  resulted  from  butter.  Probably  the  longevity 
of  B.  typhosus  in  butter  would  vary  greatly  with  the  temperature 
and  other  factors,  but  it  is  quite  evident  that  butter  should  not  be 
produced  from  infected  milk. 

Tubercle  bacilli  multiply  only  slowly— if  at  all— in  milk;  hence, 
it  is  the  initial  contamination  which  counts.  But  we  have  seen  in 


388 


BACTERIA  IN  OTHER  FOODS 


the  preceding  chapter  that  these  organisms  often  find  their  way  into 
milk,  and  the  following  table  after  Briscoe  and  MacNeal  indicates 
that  they  often  occur  in  butter. 

INCIDENCE   OF  TUBERCLE   BACILLI   IN  MARKET   BUTTER. 


Author. 

Date. 

Place. 

iSamples 
exam- 
ined. 

Samples 
positive. 

Percent, 
positive. 

Remarks. 

Brusaferro 

1890 

Turin 

9 

1 

11.1 

Roth    .      .      . 

1894 

Zurich 

20 

2 

10.0 

Microscopic  method. 

Obermuller      . 

1895 

Berlin 

13 

8 

61.0 

Schuchardt 

1896 

Marburg 

42 

0 

0.0 

Obermuller     . 

1897 

Berlin 

14 

14 

100.0 

16  tested,  2  lost. 

Groning     . 

1897 

Hamburg 

17 

8 

47.0 

Himesch    . 

1897 

Wien 

? 

0 

0 

Rabinowitsch  . 

1897 

Berlin 

30 

0 

0 

Rabinowitsch  . 

1897 

Philadelphia 

50 

0 

0 

Petri    .      .      . 

1897 

Berlin 

102 

33 

32.3 

Hormon  and 

Morgenroth 

1897 

Berlin 

10 

3 

30.0 

Rabinowitsch  . 

1899 

Berlin 

15 

2 

13.3 

First  series. 

Rabinowitsch  . 

1899 

Berlin 

? 

? 

87.2 

Second  series. 

Rabinowitsch  . 

1899 

Berlin 

15 

15 

100.0 

Third  series. 

Rabinowitsch  . 

1899 

Berlin 

19 

0 

0 

Fourth  series. 

Obermuller      . 

1899 

Berlin 

10 

4 

40.0 

Korn    . 

1899 

Freiburg 

17 

4 

23.5 

Ascher. 

1899 

Konigsburg 

27 

2 

7.4 

Jager   . 

1899 

Konigsburg 

3 

1 

33.3 

Coggi  . 

1899 

Milan 

100 

12 

12.0 

Weissenfield    . 

1899 

Bonn 

32 

3 

9.4 

Grassberger    , 

1899 

Wien 

10 

0 

0 

Herbert     .      . 

1899 

Tubingen 

43 

0 

0 

Herbert     .      . 

1899 

Wurttem- 

58 

0 

0 

Pseudotuberculosis, 

burg 

5  per. 

Herbert     .      . 

1899 

Berlin 

20 

0 

0 

Pseudotuberculosis, 

8  per. 

Herbert     .      . 

1899 

Munchen 

5 

0 

0 

Pseudotuberculosis, 

4  per. 

Abenhausen    . 

1900 

Marburg 

39 

0 

0 

Hellstrom 

1900 

Helsingfors 

8 

1 

12.5 

12  samples,  4  lost. 

Bomhoff    .      . 

1900 

Marburg 

28 

0 

0 

39  samples,  11  lost. 

Pawlowsky 

1900 

Kiew 

23 

1 

4.3 

Tobler       .      . 

1901 

Zurich 

12 

2 

16.7 

Lorenz 

1901 

Dorpat 

30 

0 

0 

Markl        .      . 

1901 

Wien 

43 

0 

0 

Heir  and 

Beninde       .      1901 

Breslau 

52 

6 

11.1 

Two  were  doubtful. 

Aujeszky  . 

1902 

Budapest 

17 

3 

17.6 

Thu      .      .      . 

1902 

Christiania 

16 

0 

0 

Teichert    .      . 

1904 

Rosen 

40 

12 

30.0 

Reitz    .      .      . 

1906 

Stuttgart 

94 

8 

8.5 

Butter  from   eighty- 

eight  dairies. 

Eber    .      .      . 

1908 

Leipzic 

150 

18 

12.0 

Briscoe  and 

MacNeal     . 

1911 

Urbana,  111. 

6 

2 

33.2 

Eber    .      .      . 

1912 

15.6 

Creamery  butter. 

Rosenau  et  al 

1914 

Boston 

21 

2 

9.4 

52   per  cent,   of    the 

samples     contained 

•  acid-fast  bacilli. 

Marchiotti 

1917    ! 

1 

25 

24.0 

BACTERIA  IN  BUTTER 


389 


Tubercle  bacilli  have  also  been  found  in  oleomargarine.  Briscoe 
and  MacNeal  tabulated  the  analysis  of  209  samples,  4.3  per  cent, 
of  which  were  found  to  contain  tubercle  bacilli. 

The  longevity  of  tubercle  bacilli  in  butter  is  even  greater  than  that 
of  B.  typhosus,  for  Schroeder  and  Col  ton  demonstrated  their  pres- 
ence in  butter  which  had  been  kept  for  one  hundred  and  sixty  days ; 


130- 
120- 
110- 
100- 
90- 
80- 
70- 
60- 
50- 

30- 
20- 
10- 


Commercial  score 
Bacteria  content 


10        20        30        40        50        60        70        80        90       100      110      120      130 

Days 

FIG.  47. — Bacteria  content  and  commercial  score  of  Cheddar  cheese.      (After  Harding 

and  Prucha,  1903.) 

and  contrary  to  prevailing  opinion  Mohler,  Washburn,  and  Rogers 
found  tubercle  bacilli  were  not  devitalized  by  cold  storage,  and  in 
salted  butter  they  were  found  to  retain  their  virulence  for  six 
months. 

Diphtheria  bacteria  have  been  found  in  butter,  and  an  outbreak 
at  Lewiston,  Minnesota,  was  believed  to  have  been  caused  by  eating 
infected  butter.  There  had  been  no  diphtheria  in  the  place  until  a 


390 


BACTERIA  IN  OTHER  FOODS 


boy  returned  from  the  "Twin  Cities"  after  an  attack  of  diphtheria. 
The  milk  from  the  farm  where  he  lived  was  sent  to  a  creamery  and 
every  family  in  the  place,  in  which  there  was  diphtheria,  was  found 
to  have  used  butter  from  this  creamery.  Experiments  have  shown 
that  the  diphtheria  bacilli  can  live  in  butter  for  a  month. 

Bacteria  in  Cheese.— If  undesirable  species  of  microorganisms  are 
present  in  the  milk  they  will  pass  into  the  cheese  and  there  produce 
their  harmful  effect.  This  is  more  important  in  cheese-making 
than  in  butter-making  since  it  has  not  been  found  possible  to 
make  many  of  the  important  varieties  of  cheese  from  pasteurized 
milk.  The  bacteria  most  dreaded  by  the  cheese  manufacturer  are 
those  of  the  B.  coli  aerogens  group.  These  give  rise  to  the  formation 
of  large  holes  which  can  be  often  taken  to  indicate  bad  flavor  as  the 
organisms  produce  in  addition  to  hydrogen  and  carbon  dioxid 
offensive  smelling  and  tasting  compounds. 

The  bacteria  of  cheese  increase  during  the  first  few  weeks  (Fig. 
45),  after  which  there  is  a  decrease,  but  even  in  ripened  cheese  there 
are  millions  of  bacteria. 

Pathogenic  bacteria  which  are  in  the  milk  will  find  their  way  into 
the  cheese,  as  Schroeder  and  Brett  purchased  256  sample  s  of  cheese 
on  the  Washington  market  and  examined  them  for  tubercle  bacilli 
by  means  of  guinea-pig  inoculation;  7.42  per  cent,  contained  tubercle 
bacilli.  Probably  many  of  the  tubercle  bacilli  die  during  the  ripening 
process,  but  this  cannot  be  entirely  depended  upon,  as  Washburn 
and  Done  prepared  a  cheese  from  infected  milk  and  after  220  days 
produced  generalized  tuberculosis  by  injection  into  guinea-pigs, 
and  even  after  260  days  injection  of  emulsions  caused  slight  lesions. 
According  to  the  findings  of  Rowland,  B.  typhosus  and  M.  cholera 
soon  perish  in  cheese. 

Bacteria  in  Ice  Cream.— The  number  of  bacteria  in  ice  cream  may 
at  times  be  enormous.  There  is  only  a  slight  decrease  on  storage, 
as  is  seen  from  the  following  results  of  Hammer  and  Goss : 

SHOWING  THE  EFFECT  OF  STORAGE  ON  THE  NUMBER  OF  BACTERIA  IN 

ICE   CREAM. 


Mixed 

236,000 

32,800,000 

120,000 

172,500,000 

110,000,000 

120,000,000 

Frozen 

735,000 

30,850,000 

146,000 

271,000,000 

170,000,000 

140,000,000 

Days  old: 

1  .   . 

360,000 

7,750,000 

137,000 

157,000,000 

194,000,000 

70,000,000 

2  .   . 

310,000 

4,450,000 

216,000 

128,000,000 

216,000,000 

71,000,000 

3  .   . 

260,000 

2,435,000 

52,000,000 

102,000,000 

4  .   . 

1,150,000 

152,000 

5  .   . 

310,000 

300,000 

34,000,000 

39,000,000 

41,000,000 

6  .   . 

139,000 

7  .   . 

156,000 

31,000,000 

54,000,000 

8  .   . 

.  . 

61,000,000 

9  .   . 

36,000,000 

10 

11 

1  ft 

15,000,000 

BACTERIA  IN  CONDENSED  MILK  391 

Freezing  does  not  kill'' the  bacteria  in  milk  or  cream;  hence,  ice 
cream  may  and  does  convey  all  of  the  milk-borne  diseases.  Further- 
more, the  ice  cream  is  often  made  in  unsanitary  places  and  handled 
in  an  unsanitary  manner;  so  that  even  though  the  milk  be  safe 
there  are  numerous  opportunities  for  infection,  especially  where  ice 
cream  is  vended  on  the  street  or  from  the  little  ice-cream  stands  on 
the  corner. 

Bolton  reported  experiments  on  the  freezing  of  B.  typhosus  in 
cream.  One-twentieth  of  the  organisms  were  alive  after  one  month 
and  even  after  forty-five  days  some  of  the  organisms  were  alive. 
Furthermore,  epidemics  of  typhoid  have  actually  been  traced  to 
ice  cream.  Gumming  reported  23  cases  which  developed  among 
twenty-nine  persons  who  partook  of  ice  cream  at  a  school  picnic 
at  Helm,  California,  in  1916.  Ice  cream  was  the  only  food  partaken 
of  by  all,  and  as  chocolate  ice  crream  was  the  favorite  flavor  this 
was  determined  to  be  the  source  of  the  infection.  This  was  because 

(1)  those  not  partaking  of  it  did  not  become  ill,  (2)  those  partaking 
of  it  but  no  other  food  were  taken  ill,  (3)  those  eating  chocolate  ice 
cream  were  taken  with  acute  intestinal  symptoms,  and  (4)  those 
eating  the  largest  quantity  of  chocolate  ice  cream  were  the  most 
seriously  ill. 

Dysentery  is  also  often  spread  by  means  of  ice  cream.  Smillie 
studied  75  cases  and  found  the  etiology  of  them  to  be  as  follows: 

Cases. 

Contact    with  an  acute  case 21 

Contact  with  a  carrier 2 

Contact  with  house  cases 4 

Condensed  milk  epidemic 15 

Ice-cream  cones 9 

Flies 6 

Milk 1 

Water 1 

Fruit 1 

Unknown 15 

The  dysentery  bacillus  of  Flexner  was  actually  isolated  from  the 
ice-cream  cones. 

Hamilton  has  pointed  out  that  ice-cream  epidemics  can  be  pre- 
vented by  (1)  the  use  of  ingredients  with  a  clean  sanitary  history, 

(2)  the  use  of  properly  cleaned  utensils  and  a  clean  factory,  anfl  (3) 
the  proper  handling  of  materials  by  individuals  with  a  clean  bill 
of  health.    The  first  of  these  is  to  be  controlled  by  the  pasteurization 
of  the  milk  and  cream;  the  second  by  frequent  inspection;  and  the 
last  requires  regular  and  careful  inspection  of  all  workers  for  com- 
municable diseases. 

Bacteria  in  Condensed  Milk.— Sweetened  condensed  milk  is  not 
intended  to  be  sterile.  The  large  quantity  of  sugar  added  prevents 
the  growth  of  microorganisms.  But  the  unsweetened  or  evaporated 


392  BACTERIA  IN  OTHER  FOODS 

milk  is  sterilized  after  sealing,  and  hence  when  properly  done  the 
finished  product  should  contain  no  bacteria.  The  organisms  which 
at  times  survive  pasteurization  and  later  may  cause  spoilage  are 
B.  subtilis,  11.  mesentericus,  and  B.  coagulant. 

The  degree  of  heat  to  which  all  the  condensed,  concentrated,  and 
powdered  milk  are  subjected  is  probably  sufficient  to  kill  all  tubercle 
bacilli  and  typhoid  bacilli,  but  Andrews'  work  would  indicate  that 
at  times  condensed  milk  may  act  as  a  differential  medium  for 
Staphylococci.  He  reports  instances  where  at  time  of  condensation 
a  few  Staphylococci  Pyogenes  aureus  were  present,  but  later  when  the 
cans  were  opened  many  were  present. 

Bacteria  in  Bread.— The  interior  of  the  loaf  reaches  a  temperature 
of  101°  to  103°  C.  and  the  crust  125°  to  140.5°  C.  in  the  baking 
process;  hence  only  the  more  resistant  sporebearing  organisms 
would  survive  the  baking  process.  Disease  germs,  therefore,  seldom 
—if  ever—  occur  in  the  freshly  baked  bread.  However,  B.  mesen- 
tericus vulgatm,  and  probably  other  organisms  which  cause  slimy 
or  ropy  bread,  may  survive  and  cause  considerable  economic  loss. 

When  they  have  found  their  way  into  a  bakery  the  ease  with 
which  they  are  overcome  depends  upon  whether  they  are  in  the 
yeast,  on  the  utensils,  or  in  the  flour.  If  they  are  in  the  yeast,  a  new 
start  must  be  obtained,  and  Kayser  suggests  the  use  of -acidulated 
water  for  washing  all  of  the  apparatus  and  even  states  that  some  of 
the  apparatus  may  have  to  be  discarded.  Great  economic  loss, 
however,  results  when  the  flour  is  the  infected  material. 

Although  bread  is  free  from  pathogenic  bacteria  when  it  leaves 
the  oven,  this  is  often  not  true  when  it  reaches  the  consumer,  for 
unwrapped  bread  must  ever  remain  a  constant  danger.  But  when 
once  wrapped,  the  danger  is  not  as  great,  for  the  wrapper  acts  as  a 
protection  and  if  carelessly  handled  tells  the  tale  to  such  an  extent 
that  it  may  be  refused  by  the  consumer. 

Bacteria  in  Eggs.— All  investigators  have  found  more  bacteria 
in  the  egg  yolk  than  in  the  egg  white,  and  many  of  those  who  have 
found  no  bacteria  in  egg  white  have  assumed  that  this  part  of  the 
egg  possesses  a  bactericidal  action.  Rettger  considers  that  the 
contents  of  normal  fresh  eggs  are  as  a  rule  sterile,  although  he  con- 
siders it  quite  probable  that  an  egg  yolk  may  become  invaded  before 
it  is  expelled  from  the  ovary.  But  this  he  considers  an  uncommon 
occurrence,  except  when  the  ovary  is  infected  with  the  organism 
of  bacillary  white  diarrhea. 

The  percentage  of  infected  eggs  found  by  different  investigators 
as  reported  by  Tanner  is  given  below: 

Number  Per  cent. 

Author.  examined.  infected. 

Rettger ' 3516                           9.5 

Rettger  (10  c.c.  samples) 647  3.86 

Bushnell  and  Mauer 27.59  23.70 

Mauer 600  18.10 

Hadley  and  Caldwell      .  2520  8.70 


BACTERIA  IN  MEAT  393 

The  kind  of  organism'' and  the  number  of  times-found  by  Rettger 
are  listed  below : 

Fresh  eggs.  Number  of  times 

found. 

Staphylococcus,  usually  aureus  and  albus 74 

Subtilis  group,  usually  B.  mesentericus  and  B.  ramosus       ....  60 

B.  coli  and  closely  related  organisms 43 

Proteus  group 30 

Streptococcus 14 

Micrococcus  (tetragenus,  etc.)        9 

Streptothrix 6 

Diphtheroid  bacillus 5 

Putrefactive  anaerobes 5 

B.  fluorescens 2 

Mold "...  4 

B.  mucosus 3 

Mixed 2 

Total 257 

Hadley  and  Caldwell  studied  forty  different  strains  isolated  from 
eggs.  They  were  divided  as  follows:  rods,  28;  cocci,  11 ;  spirillum,  1. 
They  found  no  member  of  the  hemorrhagic  septicemia,  intestinal, 
proteus  colon,  enteritidis,  typhoid,  dysentery,  nor  diphtheria  group. 

Rettger  considers  that  under  normal  conditions  the  shell  is  bac- 
terium-proof. Moisture  lessens  its  impervious  character,  however, 
and  when  combined  with  dirt  or  filth  makes  it  possible  for  micro- 
organisms to  enter  and  bring  about  decomposition.  Hence,  eggs 
should  be  stored  under  sanitary  conditions. 

Cold  storage,  frozen,  and  dried  eggs  often  contain  millions  of 
bacteria,  yet  of  all  food,  so  far  as  known,  eggs  are  less  liable  to  con- 
tain harmful  products  or  to  convey  disease  than  any  other  single 
food  of  animal  origin.  They  have  an  exceptionally  clean  health 
record .  There  is  no  known  infection  of  the  hen  transmissible  through 
the  eggs  to  man.  The  literature  is  exceptionally  free  from  instances 
where  sickness  has  been  attributed  to  eggs  except  in  the  case  of 
anaphylaxis  which  is  undoubtedly  due  to  an  idiosyncrasy  of  the 
individual  and  not  to  any  inherent  injurious  constituent  of  the  egg. 

Bacteria  in  Meat.— It  is  usually  considered  that  the  tissues  of 
healthy  animals  are  free  from  bacteria,  but  H#agland  states  that 
certain  bacteria  (chiefly  micrococci)  may  be  normally  present  in  the 
carcass  of  healthy  animals  slaughtered  for  beef.  These  bacteria  he 
considers  possess  no  pathological  significance  and  do  not  appear  to 
multiply  in  the  cold-stored  carcasses,  provided  the  cold  storage 
room  is  maintained  at  the  proper  temperature. 

Meat  kept  for  some  time  may  contain  many  bacteria.  Weinzirl 
and  Newton  found  that  four  out  of  ten  samples  of  meat  which  had 
been  stored  at  —10°  C.  for  one  year,  contained  over  10,000,000. 

Chopped  meats  invariably  contain  many  bacteria  for  the  reason 
that  meat  used  for  that  purpose  is  often  that  which  has  been  dis- 


394  BACTERIA  JN  OTHER  FOODS 

carded  for  other  purposes  and  then  the  hashing  carries  the  bacteria 
throughout  the  mass  which  is  an  excellent  medium  for  'their  multi- 
plication. This  is  recognized  by  the  fact  that  Weinzirl  and  Newton 
proposed  a  bacterial  standard  of  not  over  10,000,000  per  grain  for 
hamburger  and  then  found  that  50  per  cent,  of  the  samples  examined 
by  them  had  to  be  condemned. 

Sausage  always  contains  numerous  bacteria,  but  as  pointed  out 
by  Carey  the  kind  of  organism  present  is  more  important  than  the 
number.  He  isolated  the  following  organisms  from  34  samples  of 
sausage  purchased  on  the  market  in  Chicago: 

Bacillus  coli        .      .      .      ...      .'    • .      .    '.      ..    .',  .    .      .  .  .  30 

Proteus  vulgaris ..:»..  .  .  11 

B.  paracolon .      .      ....'.      .      .  .  .  9 

B.  fecalis       . : 8 

Yeast       .      .      .    •.      ...      .      .      .      .      .      .  *.    •';...   . .:  ...  .  8 

Streptococcus f     .  5 

Staphylococcus  aureus    .      .      .      ....      .      .      .      .      .  .  .  2 

Bacteria  in  Canned  Foods.— The  majority  of  the  canned  meats 
and  fruits  are  free  from  bacteria,  but  in  the  case  of  swelled  and 
spoiled  products  numerous  organisms  are  found. 

Some  of  the  organisms  identified  by  Donk  as  causing  spoilage  of 
canned  goods  were  as  follows : 

M.  acidi  in  cheese. 

M.  candicans  in  roast  beef,  sardines,  and  bulk  granulated  sugar. 

M.  candidus  in  baked  beans. 

M .  cereus  in  baked  beans. 

M.  lactis  in  cheese. 

M.  luteusin  corn. 

M.  pyogenes  in  two  samples  of  Maine  style  corn  and  one  sample 
of  canned  corn  on  cob. 

M .  stellatus  in  canned  roast  beef. 

B.  cloacce  in  canned  roast  beef. 

B.  detrudens  in  cheese. 

B.  licheniformis  in  stringbeans. 

B.  megatherium  in  sauerkraut  brine  (not  canned)  and  cheese. 

B.  mesentericus  in  cheese. 

B.  pammellii  in  cheese. 

B.  subtilis  in  corn. 

B.  tenus  in  cheese. 

B.  viscosus  in  cheese. 

B.  xfilatis  in  spinach  and  bulk  granulated  sugar. 

B.  welchii  in  corn. 

B.  mdgatus  in  two  samples  of  corn. 

REFERENCES. 

Rosenau:     Preventative  Medicine  and  Hygiene. 
Tanner:     Bacteriology  and  Mycology  of  Foods. 


CHAPTER   XXXIV. 
BACTERIA  AND  FOOD-POISONING. 

THE  term  "food-poisoning,"  or  ptomain  poisoning,  in  the  past 
has  been  used  to  cover  a  multitude  of  physiological  disturbances. 
As  Jordan  points  out,  "that  convenient  refuge  from  etiological 
uncertainty  '  ptomain  poisoning'  is  a  diagnosis  that  unquestion- 
ably has  been  made  to  cover  a  great  variety  of  diverse  conditions 
from  appendicitis  and  pain  caused  by  gall-stones  to  the  simple 
abdominal  distention  resulting  from  reckless  gorging."  But  even 
when  account  is  taken  of  this,  its  toll  of  human  life  and  suffering  is 
great.  Food-poisoning  is  also  a  great  cause  of  inefficiency,  depres- 
sion, sluggish  mental  processes,  dissatisfaction,  or  abnormal  irrita- 
bility which  are  often  overlooked  or  attributed  to  other  causes. 

Classes  of  Food-poisoning.— Present  knowledge  permits  the  follow- 
ing rough  classification  of  food-poisoning : 

1.  Poisoning  due  to  the  eating  of  foods  which  naturally  contain 
poisonous  products. 

2.  Poisoning  due  to  the  eating  of  foods  containing  mineral  poisons 
added  either  intentionally  or  accidentally. 

3.  The  eating  of  foods  which  are  normally  non-poisonous  but 
which  have  been  obtained  from  animals  suffering  from  disease. 

4.  The  eating  of  food  which  has  been  accidentally  infected  with 
pathogenic  bacteria  in  handling  or  preparation. 

5.  The  eating  of  foods  which  contain  poisonous  products  of 
bacterial  katabolism-toxins. 

6.  The  eating  of  a  normal  food  by  an  individual  who  possesses 
peculiar  idiosyncrasies  toward  a  specific  food. 

Poisonous  Foods.— The  first  group  consists  of  naturally-occurring 
plants  and  animals  which  are  always  poisonous  or  become  so  during 
certain  seasons  of  the  year.  According  to  Chestnut  there  are 
16,673  leaf-bearing  plants  included  in  Heller's  Catalog  of  North 
American  Plants.  Of  these  nearly  500  have  been  alleged  to  be 
poisonous,  but  fortunately  only  a  few  are  ever  accidentally  par- 
taken of  by  man.  Chestnut  lists  about  thirty  important  poisonous 
plants  occurring  in  the  United  States  and  some  of  these  are  not 
known  to  be  poisonous  except  to  domestic  animals.  Some  of  the 
more  common  are  as  follows: 

American  false  hellebore  (Veratrum  viridi)  —mistaken  for  marsh- 
marigold. 


396  BACTERIA  AND  FOOD-POISONING 

Kentucky  coffee  tree  (Gymnodadus  diowd}  — mistaken  for  honey- 
locust. 

Broad  leaf  laurel  (Kalmia  litifolia)—  mistaken  for  wintergreen. 

Water  hemlock  (Cicuta  maculata)  —  roots  mistaken  for  horse- 
radish, artichoke,  parsnip,  etc. 

By  far  the  most  common  of  these  cases  of  plant-poisoning  is  due 
to  the  eating  of  the  poisonous  mushrooms  or  "  toadbtools"  (Amanda 
muscaria),  (A.  phalloides),  (A.  verna). 

The  symptoms  of  poisoning  with  A.  phalloides  is  thus  described 
by  Ford: 

"  Following  the  consumption  of  the  fungi  there  is  a  period  of  six 
to  fifteen  hours  during  which  no  symptoms  of  poisoning  are  shown 
by  the  victims.  This  corresponds  to  the  period  of  incubation  of 
other  intoxications  or  infections.  The  first  sign  of  trouble  is  sudden 
pain  of  the  greatest  intensity  localized  in  the  abdomen,  accompanied 
by  vomiting,  thirst,  and  choleraic  diarrhea  with  mucous  and  bloody 
stools.  The  latter  symptom  is  by  no  means  constant.  The  pain 
continues  in  paroxysms  often  so  severe  as  to  cause  the  peculiar 
Hippocratic  f acies,  la  face  vulteuse  of  the  French,  and  though  some- 
times ameliorated  in  character,  it  usually  recurs  with  greater 
severity.  The  patients  rapidly  lose  strength  and  flesh,  their 
complexion  assuming  a  peculiar  yellow  tone.  After  three  to  four 
days  in  children  and  six  to  eight  in  adults  the  victims  sink  into  a 
profound  coma  from  which  they  cannot  be  roused  and  death  soon 
ends  the  fearful  and  useless  tragedy.  Convulsions  rarely  if  ever 
occur  and  when  present  indicate,  I  am  inclined  to  believe,  a  mixed 
intoxication,  specimens  of  Amanita  muscaria  being  eaten  with  the 
phalloides.  The  majority  of  individuals  poisoned  by  the  '  deadly 
Amanita'  die,  the  mortality  varying  from  60  to  100  per  cent,  in 
various  accidents,  but  recovery  is  not  impossible  when  small 
amounts  of  the  fungus  are  eaten,  especially  if  the  stomach  be  very 
promptly  emptied,  either  naturally  or  artificially." 

Metallic  Poisons.— Various  canned  goods  have  been  repeatedly 
accused  of  causing  poisoning,  but  the  cases  in  which  this  has  occurred 
when  the  foods  have  been  sterilized  by  the  pressure  method  are 
extremely  rare.  Where  it  has  caused  trouble  it  is  usually  due  to 
some  metallic  poison  found  in  the  cans  and  not  to  poisons  developed 
in  the  food  due  to  bacterial  activity. 

Asparagus  is  often  looked  upon  as  one  of  the  canned  products 
most  likely  to  cause  poisoning.  This  is  due  in  a  large  measure  to  the 
fact  that  asparagus  takes  up  large  quantities  of  tin,  and  some  indi- 
viduals are  especially  susceptible  to  this  substance.  The  quantity 
of  tin,  and  especially  copper,  which  is  taken  up  in  most  cases  varies 
with  the  amount  and  kind  of  acid  found  in  the  fruit  or  vegetables. 
Moreover,  when  a  low  or  poor  grade  of  copper  is  used,  it  is  more 
readily  attacked  by  the  fruits  than  are  the  pure  compounds. 


TYPICAL  PARATYPHOID  OUTBREAKS  397 

Fairly  large  quantities  of  copper  have  to  be  eaten  before  death 
results,  and  it  is  doubtful  whether  many  foods  would  dissolve 
sufficient  to  result  fatally.  Whereas  a  small  small  quantity  of  one 
of  the  metallic  poisons  taken  once  may  cause  no  ill  effects,  their 
constant  use  would,  for  their  action  is  cumulative.  Moreover, 
sanitarians  insist  that  chemical  substances  likely  to  be  irritating  to 
the  human  tissues  in  assimilation  or  elimination  should  not  be 
employed  in  food.  Each  new  irritant,  even  in  small  quantities, 
may  add  to  the  burden  of  organs  already  weakened  by  age  or 
previous  harsh  treatment. 

Animals  Suffering  from  Disease.— The  milk  and  flesh  of  animals 
suffering  with  certain  diseases  are  continually  being  used  without 
adequate  cooking,  the  result  being  that  thousands  die  each  year  from 
this  cause.  The  majority  of  these  cases  come  from  the  use  of  milk, 
which  has  been  considered  in  an  earlier  chapter.  A  typical  out- 
break of  paratyphoid  due  to  the  eating  of  diseased  meat  is  thus 
given  by  Jordan : 

"The  most  characteristic  examples  of  'food  poisoning,'  popularly 
speaking,  are  those  in  which  the  symptoms  appear  shortly  after 
eating  and  in  which  gastro-intestinal  disturbances  predominate. 
In  the  typical  group-outbreaks  of  this  sort  all  grades  of  severity 
are  manifested,  but  as  a  rule  recovery  takes  place.  The  great 
majority  of  such  cases  that  have  been  investigated  by  modern 
bacteriological  methods  show  the  presence  of  bacilli  belonging  to  the 
so-called  paratyphoid  group  (B.  paratyphosus  or  B.  enteritidis) . 
Especially  is  it  true  of  meat-poisoning  epidemics  that  paratyphoid 
bacilli  are  found  in  causal  relation  with  them.  Hiibener  enumerates 
forty-two  meat-poisoning  outbreaks  in  Germany  in  which  bacilli  of 
this  group  were  shown  to  be  implicated,  and  Savage  gives  a  list  of 
twenty-seven  similar  outbreaks  in  Great  Britain.  In  the  United 
States  relatively  few  outbreaks  of  this  character  have  been  placed 
on  record,  but  it  cannot  be  assumed  that  this  is  due  to  their  rarity, 
since  no  adequate  investigation  of  food-poisoning  cases  is  generally 
carried  out  in  our  American  communities. 

"  Typical  Paratyphoid  Outbreaks.— Kaensche  describes  an  outbreak 
at  Breslau  involving  over  eighty  persons  in  which  chopped  beef  was 
apparently  the  bearer  of  infection .  The  animal  from  which  the  meat 
came  had  been  ill  with  sever  diarrhea  and  high  fever  and  was 
slaughtered  as  an  emergency  measure  (notgeschlachtet) .  On  exami- 
nation a  pathological  condition  of  the  liver  and  other  organs  was 
noted  by  a  veterinarian  who  declared  the  meat  unfit  for  use  and 
ordered  it  destroyed.  It  was,  however,  stolen,  carried  secretly 
to  Breslau,  and  portions  of  it  were  distributed  to  different  sausage- 
makers,  who  sold  it  for  the  most  part  as  hamburger  steak  (Hack- 
fleisch) .  The  mea^t  itself  presented  nothing  abnormal  in  color,  odor, 
or  consistency.  Nevertheless,  illness  followed  in  some  cases  after 


398  BACTERIA  AND  FOOD-POISONING 

the  use  of  very  small  portions.  With  some  of  those  affected  the 
symptoms  were  very  severe,  but  there  were  no  deaths.  Bacilli  of 
the  Bacillus  enteritidis  type  were  isolated  from  the  meat. 

"A  large  and  unusually  severe  outbreak  reported  by  McWeeney 
occurred  in  November,  1908,  among  the  inmates  of  an  industrial 
school  for  girls  at  Limerick,  Ireland.  There  were  73  cases  with 
9  deaths  out  of  the  total  number  of  197  pupils.  The  brunt  of  the 
attack  fell  on  the  first  or  Senior  class  comprising  67  girls  between 
the  ages  of  thirteen  and  seventeen.  Out  of  55  girls  belonging  to  this 
class  who  partook  of  beef  stew  for  dinner  53  sickened,  and  8  of  these 
died.  One  of  the  two  who  were  not  affected  ate  the  gravy  and 
potatoes  but  not  the  beef.  Some  of  the  implicated  beef  was  also 
eaten  as  cold  meat  by  girls  in  some  of  the  other  classes,  and  also 
caused  illness.  Part  of  the  meat  had  been  eaten  previously  without 
producing  any  ill  effects.  'The  escape  of  those  who  partook  of 
portions  of  the  same  carcass  on  October  27  and  29  (five  days  earlier) 
may  be  accounted  for  either  by  unequal  distribution  of  the  virus  or 
by  thorough  cooking  which  destroyed  it.  Some  of  the  infective  ma- 
terial must,  however,  have  escaped  the  roasting  of  the  29th,  and 
multiplying  rapidly,  have  rendered  the  whole  piece  intensely  toxic 
and  infective  during  the  five  days  that  elapsed  before  the  fatal  Tues- 
day when  it  was  finally  consumed.'  The  animal  from  which  the  fore- 
quarter  of  the  beef  was  taken  had  been  privately  slaughtered  by  a  local 
butcher.  No  reliable  information  could  be  obtained  about  the 
condition  of  the  calf  at,  or  slightly  prior  to,  slaughter.  The  meat, 
however,  was  sold  at  so  low  a  price  that  it  was  evidently  not  regarded 
as  of  prime  quality.  In  this  outbreak  the  agglutination  reactions 
of  the  blood  of  the  patients  and  the  characteristics  of  the  bacilli 
isolated  showed  the  infection  to  be  due  to  a  typical  strain  of  Bacillus 
enteritidis." 

Offending  Foods.— Meat  is  so  often  the  cause  of  poisoning  that 
the  terms  ."  meat-poisoning"  and  food-poisoning"  have  come  to 
be  used  almost  synonymously.  Of  meats,  chicken  and  pork  are 
more  likely  to  cause  poisoning  than  are  meats  from  other  animals, 
while  the  internal  organs— liver  and  kidney— are  more  likely  to 
Contain  disease-producing  bacteria  than  are  the  muscular  tissues. 
Sausages,  hamburger  steaks,  meat  pies,  puddings,  and  jellies  are 
especially  likely  to  cause  food-poisoning.  This  is  probably  due  to 
the  products  from  which  they  are  made,  the  methods  of  treating,  and 
the  fact  that  the  heat  used  in  cooking  such  foods  is  not  sufficient  to 
kill  the  bacteria  in  the  food.  While  there  are  a  few  cases  on  record 
where  individuals  have  been  poisoned  by  the  eating  of  freshly  well- 
cooked  meats  they  are  so  rare  as  to  be  of  little  importance:  so  the 
thorough  cooking  of  meat  greatly  diminishes  the  likelihood  of 
trouble. 


HUMAN  INFECTION  399 

Human  Infection.— It  is  necessary  that  food  be  protected  from 
contamination  during  the  whole  process  of  preparation  and  serving 
to  prevent  its  infection  with  pathogenic  bacteria,  as  is  illustrated  by 
the  remarkable  instance  reported  by  Sawyer  where  ninety-three 
typhoid  cases  were  caused  by  eating  Spanish  spaghetti  served  at  a 
public  dinner.  Investigation  showed  that  the  dish  had  been 
prepared  by  a  woman  typhoid-carrier  who  was  harboring  living 
typhoid  bacilli  at  the  time  she  prepared  the  dish.  The  dish  was 
baked  after  it  was  infected,  but  the  baking  was  shown  by  laboratory 
experiments  to  have  incubated  the  bacteria  instead  of  sterilizing 
the  food. 

Then  there  is  the  celebrated  case  of  Typhoid  Mary,  investigated 
by  Saper.  In  the  pursuit  of  her  work  as  a  cook  in  and  about  New 
York  City  she  is  known  to  have  caused  at  least  seven  typhoid  out- 
breaks in  various  families  and  one  extensive  hospital  epidemic. 

The  danger  from  this  source  is  voiced  by  Chapin  as  follows: 
"There  are  doubtless  200,000  cases  of  this  disease  (typhoid  fever) 
in  the  United  States  each  year.  If  only  3  per  cent,  of  these  become 
chronic  carriers,  and  if  a  carrier  remains  such  only  three  years,  we 
should  have  a  carrier  population  of  18,000  persons,  practically 
unknown  and  taking  no  precautions  against  infecting  others.  If  we 
add  to  these  the  25  per  cent,  of  convalescents,  who  for  some  weeks 
are  excreting  the  bacilli  in  their  urine,  it  appears  that  there  is  a  very 
respectable  army  of  unrecognized  sources  of  typhoid  infection." 

This  is  a  situation  which  will  be  solved  only  when  all  handlers 
of  food  in  public  places  are  examined  for  various  diseases  which  are 
transmissible  through  food.  A  move  in  the  right  direction  has  been 
made  by  the  California  State  Board  of  Health  which  enters  into  the 
following  agreement  with  all  carriers  discovered: 

"  I  have  this  day  been  informed  that  my  excreta  contain  typhoid 
bacilli  and  that,  unless  unusual  precautions  are  taken,  persons  will 
contract  the  infection  from  me.  Realizing  this  danger  I  agree  to 
observe  the  precautions  stated  below,  and  request  that  I  be  permitted 
to  remain  in  free  communication  with  other  persons. 

"1.  I  will  take  no  part  in  the  preparation  or  handling  of  food 
which  will  be  consumed  by  persons  outside  of  my  immediate  family, 
and  I  will  not  participate  in  the  management  of  a  boarding  house, 
restaurant,  food  store,  or  in  any  other  occupation  involving  the 
preparation  or  handling  of  food. 

"2.  I  will  not  dispose  of  my  excretions  in  a  toilet  to  which  flies 
have  access  without  first  exposing  such  excretions  to  either  a  5  per 
cent  dilution  of  liquor  formaldehyd  or  5  per  cent,  phenol  (carbolic 
acid). 

"3.  I  will  notify  the  local  health  officer  of  any  cases  of  typhoid 
among  persons  with  whom  I  come  in  contact. 

"4.  I  will  inform  the  local  health  officer  of  any  contemplated 


400  BACTERIA  AND  FOOD-POISONING 

change  of  residence  so  that  he  can  notify  the  State  Board  of  Health 
and  obtain  its  approval. 

"5.  I  will  submit  specimens  for  examination  when  requested  by 
the  State  Board  of  Health. 

"6.  I  will  fill  out  the  following  report  blank  when  submitted  to  me 
semi-annually,  and  return  the  same  to  the  California  State  Board 
of  Health: 

'  I  have,  during  the  last  six  months,  complied  to  the  best  of  my 
knowledge  with  the  five  separate  agreements  entered  into  between 
myself  and  the  California  State  Board  of  Health.  Precautions 
involved  in  these  separate  agreements  are  for  the  purpose  of  prevent- 
ing typhoid  infection.' ' 

Ptomain  Poisoning.— Plant  and  animal  tissues  under  appropriate 
temperature,  moisture,  and  aeration  putrefy.  The  proteins  are 
broken  down  with  the  formation  of  basic,  often  highly  toxic,  sub- 
stances spoken  of  as  "animal  alkaloids"  or  ptomains.  These 
compounds  are  not  poisonous  in  every  case.  The  presence  of  oxygen 
in  the  compound  seems  to  be  necessary  for  the  development  of  strong 
toxicity.  In  putrefying  mixtures  these  toxic  bodies  appear  on  or 
about  the  fifth  to  seventh  day  after  putrefaction  sets  in  and  disappear 
through  further  cleavage  more  or  less  rapidly  yielding  less  complex 
nitrogenous  substances  that  are  non-toxic.  It  was  formerly  thought 
that  they  played  a  very  important  part  in  food-poisoning.  But 
recent  work  has  indicated  that  they  are  seldom  the  causative  agent. 
\  aughan  and  Navy,  who  have  made  an  extensive  study  of  ptomains, 
have  proposed  a  very  elaborate  nomenclature  for  supposed  food- 
poisoning  due  to  their  ingestion.  Some  of  them  are  as  follows: 

Cheese-poisoning     .      .      .      ,      . Tyrotoxismus 

Fish-poisoning    .          .  .  '  ..     .      .      .    ' .      ....  Ichthyotoxismus 

Food-poisoning.      .      .      .      ......      .      .  Bromototoxismus 

Meat-poisoning        .      .      .      .....      .      .      .  Kreatoxismus 

Milk-poisoning Galactotoxismus 

Botulism.— Botulism  results  from  the  eating  of  food  in  which  the 
Bacillmbotulinus  has  grown  and  elaborated  a  poison.  The  organism 
is  a  large  bacillus  4  to  6/i  by  0.9  to  1.2/*,  having  slightly  rounded  ends 
and  they  may  arrange  themselves  in  pairs,  end  to  end,  or  in  an 
unfavorable  environment  in  long  chains.  It  is  a  strict  anaerobe,  but 
may  grow  under  imperfect  anaerobic  conditions  if  in  symbiosis  with 
certain  aerobic  bacteria.  The  optimum  temperature  for  the  growth 
of  the  bacillus  and  for  the  elaboration  of  the  poison  is  between  20° 
and  30°  C.  The  vegetative  cells  are  easily  destroyed  by  heat,  but 
the  spores  are  quite  resistant  (according  to  Van  Ermengen,  85°  C. 
for  fifteen  minutes),  but  Dickson  considers  them  even  more  resistant. 
Thorn  and  associates  isolated  strains  from  asparagus  which  survived 
steaming  under  10  pounds'  pressure  for  fifteen  minutes,  or  a  tempera- 
ture of  100°  C.  for  one  hour.  They  remain  visable,  according  to 


BOTULISM 


401 


Dickson,  for  months  or  even  years  if  protected  from  the  action  of 
light  and  air,  even  though  the  medium  in  which  they  are  immersed 
is  of  acid  reaction.  The  toxin  which  they  produce  is  quickly  de- 
stroyed by  exposure  to  the  action  of  light  and  air,  but  will  maintain 
its  virulence  for  six  months  or  more  if  kept  in  the  dark  and  sealed. 
According  to  Van  Ermengen,  the  toxicity  is  diminished  by  heating 
at  56°  C.  for  three  hours  and  destroyed  by  heating  to  80°  C.  for  half 
an  hour  or  by  boiling.  Dickson  found  that  the  toxin  may  develop 
in  mediums  such  as  green  corn,  artichokes,  asparagus,  apricots,  and 
peaches  to  which  no  traces  of  animal  proteins  have  been  added  in 
addition  to  the  various  meats. 


FIG.  48. — Bacillus  Botulinus.     (After  Dickson.) 

The  pronounced  symptoms  which  develop  on  the  ingestion  of  the 
toxin  are  thus  described  by  Wilbur  and  Ophiils: 

"  Girl,  aged  twenty-three,  Tuesday  evening,  November  23,  1913, 
ate  the  dinner  including  the  canned  stringbeans  of  the  light  green 
color  together  with  a  little  rare  roast  beef.  The  following  day  she 
felt  perfectly  normal  except  that  at  10:00  in  the  evening  the  eyes 
felt  strained  after  some  sewing.  Thursday  morning,  thirty-six 
hours  after  the  meal,  when  the  patient  awoke,  the  eyes  were  out  of 
focus,  appetite  was  not  food,  and  she  felt  very  tired.  At  night  she 
had  still  no  appetite,  was  nauseated,  and  vomited  the  noon  meal 
apparently  undigested.  Friday  morning,  two  and  one-half  days 
after  the  meal,  the  eyes  were  worse,  objects  being  seen  double  on 
quick  movement,  and  it  was  noticed  that  they  had  a  tendency  to  be 
crossed.  A  peculiar  mistiness  of  vision  was  also  complained  of. 
She  was  in  bed  until  late  in  the  afternoon,  when  she  visited  Dr. 
Black.  She  had  had  some  disturbance  in  swallowing  previous  to 
26 


402  BACTERIA  AND  FOOD-POISONING 

this  time  and  stated  that  it  felt  as  if '  something  came  up  from  below' 
that  interfered  with  deglutition.  The  fourth  day  she  remained  in 
bed,  was  much  constipated,  and  noticed  a  marked  decrease  in  the 
amount  of  urine  voided.  There  was  at  no  time  pain  except  for 
occasional  mild  abdominal  cramps,  no  headache,  subnormal  tem- 
perature, and  a  normal  pulse.  The  fourth  and  fifth  days  the  breath- 
ing became  difficult  at  times  and  swallowing  was  almost  impossible. 
The  patient  complained  of  a  dry  throat  with  annoying  thirst.  The 
sixth  day  there  were  periods  of  a  sense  of  suffocation  with  a  vague 
feeling  of  unrest  and  as  if  there  might  be  difficulty  in  getting  the 
next  breath.  The  upper  lids  had  begun  to  droop.  The  voice  was 
nasal.  When  the  attempt  was  made  to  swallow  liquids  they  passed 
back  through  the  nose.  The  patient  felt  markedly  weak. 

"  Physical  examination  at  this  time  showed  ptosis  of  both  upper 
eyelids,  dilatation  of  the  right  pupil,  sluggish  reaction  to  light  of 
both  pupils,  apparent  paralysis  of  the  internal  rectus  of  the  left 
eye,  normal  retina,  inability  to  raise  the  head,  control  apparently 
having  been  lost  of  the  muscles  of  the  neck,  inability  to  swallow, 
absence  of  taste.  The  tongue  was  heavily  coated  and  the  throat 
was  covered  with  a  viscid  whitish  mucus  clinging  to  the  mucous 
membrane.  The  soft  palate  could  be  raised  but  was  sluggish, 
particularly  on  the  right  side.  The  exudate  on  the  right  tonsil  was 
so  marked  that  it  resembled  somewhat  a  diphtheritic  membrane. 
The  seventh  day  there  was  some  change  in  the  condition;  occasional 
periods  occurred  when  swallowing  was  more  effective,  and  there  was 
less  tendency  to  strangle.  On  the  eleventh  day  there  was  some 
improvement  of  the  eyes,  still  strangling  on  swallowing,  sensation 
of  taste  was  keener,  and  the  general  condition -improved.  The 
twelfth  day  the  patient  was  able  to  move  her  head,  but  was  unable 
to  lift  it  except  when  she  took  hold  of  the  braids  of  her  hair,  and 
pulled  the  head  forward.  The  eyes  could  be  opened  slightly,  speech 
was  less  nasal  and  more  distinct,  and  improvement  in  swallowing 
was  marked.  At  the  end  of  two  weeks  the  patient  was  able  to  take 
soft  diet  freely,  and  at  four  weeks  she  was  up  in  a  chair  for  a  couple 
of  hours  complaining  only  of  general  weakness  and  inability  to  use 
her  eyes.  At  the  end  of  five  weeks  she  was  able  to  leave  the  hospital 
and  return  to  her  home  and  later  to  resume  her  regular  work." 

Prevention.— The  prevention  of  food-poisoning  from  canned  foods 
consists  of  processing  the  material  according  to  the  best  experience 
available,  the  selection  of  good,  sound  material  and  the  rejection  of 
any  infected  material.  Dirty,  wilted,  and  partly  rotted  food  carries 
many  more  organisms  into  the  canning  process  than  does  fresh, 
sound,  clean  fruits  and  vegetables.  Dirty  tables,  dirty  jars,  lids, 
and  sewage-polluted  water  and  flies  are  sources  of  contamination 
which  should  be  eliminated. 

When  a  can  presents  a  convex  appearance  (technically  called  a 


PREVENTION  403 

"  blown  can"),  or  on  opening  a  can  a  foul  smelling  gas  escapes,  it  is 
a  warning  to  the  consumer  that  the  contents  should  be  destroyed, 
not  salvaged,  fixed  up  into  salads,  mincemeat,  or  made-over  dishes 
for  human  consumption,  nor  should  it  be  fed  to  lower  animals  as 
there  are  many  cases  in  which  chickens  and  other  animals  have  been 
killed  by  such  products.  This  probably  distributes  the  organism 
on  the  premises. 

At  other  times  the  products  have  a  peculiar  rancid  odor  resembling 
spoiled  butter  which  becomes  more  pronounced  on  standing.  Such 
vegetables  should  not  be  tasted,  but  destroyed.  All  vegetables 
which  have  been  put  up  by  any  other  than  standard  methods  should 
be  boiled  before  being  eaten  or  even  tasted,  and  no  such  products 
should  be  served  as  salads  unless  they  have  been  cooked  after  remov- 
ing from  the  container. 

REFERENCES. 

Boldnau:     Food  Poisoning. 

Jordan:     Food  Poisoning. 

Tanner:     Bacteriology  and  Mycology  of  Food. 

Dickson:     Botulism  Monographs  of  the  Rockefeller  Institute  for  Medical  Research. 


CHAPTER  XXXV. 
PRESERVATION  OF  FOOD. 

WHERE  a  race  is  dependent  upon  a  local  or  seasonal  supply  of 
food  it  occurs  that  in  one  district  or  during  one  season  there  is  an 
abundance,  whereas  in  another  locality  or  at  another  season  there 
may  be  a  scarcity  which  at  times  amounts  to  a  famine.  This  state 
of  affairs  was  very  common  in  the  earlier  history  of  the  race,  but 
modern  methods  of  transportation  and  food  preservation  has  made 
it  possible  for  the  modern  individual  to  have  a  sufficient  and  varied 
diet  at  all  seasons.  A  varied  diet  is  more  certain  to  contain  all  those 
constituents  which  are  essential  to  the  body  needs  than  is  a  restricted 
or  monotonous  diet.  Moreover,  it  is  well  recognized  today  that 
nutritional  disorders  are  more  likely  to  occur  on  a  restricted  than  on 
a  varied  diet.  Hence,  the  general  result  of  the  expansion  in  the 
kinds  of  food  consumed  is  good,  but  the  food  should  be  preserved 
in  a  manner  such  that  as  little  as  possible  of  the  nutrient  constituents 
are  lost;  so  there  is  little  change  in  appearance  and  taste  and  nothing 
is  added  nor  developed  in  the  food  which  is  deleterious  to  health. 

Heat,  cold,  drying,  and  the  use  of  some  chemicals  have  long  been 
in  use  for  the  preservation  of  food,  but  it  is  only  recently  that  the 
art  has  been  developed  to  its  highest  perfection.  This  is  due  to  the 
fact  that  the  art  of  food-preserving  depends  upon  the  science  of 
bacteriology,  and  today  it  is  possible  to  preserve  some  foods  indefi- 
nitely without  injuring  their  nutritive  value  or  seriously  interfering 
with  their  taste  and  appearance,  and  all  such  methods  are  legitimate. 

But  "the  chief  harm  has  come  from  the  blind  use  of  chemical 
germicides  without  regard  for  their  harmful  properties.  The 
simplest  and  cheapest  way  to  preserve  food  is  by  adding  one  of  these 
chemicals  and  the  method  was,  therefore,  seized  upon  by  alert  men 
whose  chief  interest  was  of  the  pecuniary  kind.  The  question  was 
to  find  the  smallest  percentage  of  a  chemical  which  would  prevent  the 
decay  of  some  particular  food  product,  trusting  to  luck  that  the 
preservative  used  would  prove  harmless  to  the  consumer.  Often 
these  chemicals  were  added  with  a  liberal  hand;  further,  it  was  soon 
found  that  chemical  preservatives  could  be  used  to  preserve  food 
products  for  the  market  from  materials  already  so  decayed  as  to  be 
unsalable  in  their  original  condition." 

Methods  of  Preserving  Foods.— Methods  of  preserving  foods  may 
be  roughly  classified  as  follows: 


METHODS  OF  PRESERVING  FOODS  405 

1.  Physical  —The  more  important  methods  of  this  class  are  heat, 
cold,  and  drying.     The  last  two  are  regarded  by  the  sanitarian  as 
antiseptic  rather  than  germicidal,  as  they  usually  arrest  the  growth 
of  the  organisms,  but  do  not  kill  them. 

2.  Chemical.— Under  this  class  we  have  two  groups. 

(a)  Those   chemicals   which   preserve   through   their   influence 
upon  the  medium  in  which  they  are  placed.     The  majority  of  these 
act  by  increasing  the  osmotic  pressure  of  the  solution,  and  the 
important  constituents  used  are  salt  and  sugar.     These  are  without 
objection  from  the  hygienic  standpoint. 

(b)  Those  substances  which  bring  about  a  chemical  change  within 
the  medium  in  which  they  are  placed  or  which  combine  chemically 
with  the  living  protoplasmic  substance,  or  inhibiting  their  natural 
functions.     Their  function  is  then  to  prevent  bacterial  digestion. 
They  are  for  the  most  part  injurious,  for  if  bacteria  have  attempted 
to  digest  food  and  failed,  man  need  not  try. 

Cold.— Cold  has  come  in  recent  times  to  be  of  inestimable  value  in 
food  preservation,  and  such  food  usually  commands  a  higher  market 
value  than  food  preserved  by  other  means.  This  is  due  to  the  fact 
that  refrigerated  foods  in  most  cases  very  closely  resemble  in 
appearance,  taste,  and  nutritive  value  the  fresh  article.  Food 
can  be  kept  in  a  satisfactory  condition  in  cold  storage  for  a  very 
long  time.  The  time  varies  with  the  specific  article,  its  condition 
when  placed  in  cold  storage,  and  the  temperature  at  which  it  is  kept. 
Moreover,  some  foods  such  as  meats  pass  through  a  stage  of  ripening 
while  in  cold  storage  so  that  when  removed  they  have  developed  a 
tenderness  and  even  more  delicate  flavor  than  that  of  the  fresh 
product. 

The  temperature  at  which  foodstuffs  must  be  kept  varies  with 
different  articles.  Fish  are  usually  frozen,  dipped  in  water,  and 
refrozen.  The  formation  of  a  coating  of  ice  acts  as  a.  protection 
against  bacteria  and  also  prevents  dessication.  They  are  then 
stored  at  16°  C.  Smith  has  shown  that  fish  may  be  so  kept  for  as 
long  as  two  years  without  depreciating  in  nutritive  or  sanitary  value. 

Meats  are  usually  surface-dried  before  they  are  placed  in  cold 
storage.  This  prevents  the  formation  of  a  film  of  water  on  the 
surface  which  under  some  conditions  may  act  as  a  good  cultural 
medium.  Fggs  and  milk  are  materially  injured  by  freezing;  hence, 
they  are  ordinarily  kept  at  a  temperature  just  above  0°  C. 

Cold  is  a  disinfectant  and  not  a  germicide,  and  although  a  tempera- 
ture of  0°  C.  will  prevent  the  growth  of  pathogens,  some  saprophytes 
may  actually  multiply  at  this  temperature,  but  as  the  temperature 
is  decreased  the  pathogens  slowly  succumb.  Nevertheless,  cold 
alone  should  not  be  relied  upon  as  a  protection  against  pathogenic 
bacteria. 

Most  animal  parasites  die  If  kept  in  cold  storage  long.    Rosenau 


406  PRESERVATION  OF  FOOD 

gives  the  following  periods  for  some :  Trichinae  die  at  or  below  5°  C. 
in  twenty  days;  Taenia  saginata,  the  beef  tapeworm,  dies  in  twenty- 
one  days;  but  Taenia  solium,  the  pork  tapeworm,  may  live  more 
than  twenty-nine  days. 

Food  spoilage  in  cold  storage  is  usually  due  to  wrong  temperature. 
This  is  often  true  ot  the  home  icebox  which  is  usually  placed  so  as 
to  be  convenient,  not  considering  practicability,  and  a  survey  of 
such  refrigerators  revealed  the  fact  that  the  temperature  is  often 
15°  C.  or  higher.  Such  a  temperature  is  ideal  for  rapid  bacterial 
growth. 

Foods  taken  from  cold  storage  spoil  rapidly  as  the  bacteria  in  and 
on  the  food  have  not  been  killed  and  the  freezing  has  loosened  up  the 
texture  so  the  microorganisms  can  gain  entrance.  Moreover,  the 
enzymatic  change  which  proceeds  in  the  cold-storage  product  gives 
rise  to  substances  which  accelerate  bacterial  activity. 

Drying.— Nature's  method  of  preserving  foods  is  by  drying, 
for  this  is  the  universal  principle  used  in  preserving  seeds.  Bacteria 
must  have  moisture  to  grow  and  multiply,  and  if  the  dessication  be 
great  enough  they  die.  Pathogens  die  quite  rapidly  when  dried. 
Furthermore,  fruits,  vegetables,  and  meats  when  preserved  by  this 
method  are  usually  cooked  before  eating;  hence,  the  process  has  a 
decided  sanitary  as  well  as  economical  significance.  Although 
nothing  is  added  to  the  dried  food  and  only  water  is  lost,  yet  some 
dried  food  loses  its  savor  and  probably  at  times  decreases  in  digesti- 
bility. 

The  effectiveness  of  drying  as  a  means  of  food  preservation 
depends  upon  the  completeness  of  dessication  and  the  specific  food. 
Those  foods  which  are  rich  in  soluble  constituents  are  easily  pre- 
served by  this  method,  for  while  the  moisture  present  may  be 
considerable  yet  the  osmotic  pressure  in  the  solution  is  too  great  for 
bacterial  growth.  This  is  the  reason  grapes,  apples,  and  prunes  are 
so  easily  preserved  by  drying,  whereas  meats  and  some  fruits  are 
preserved  with  difficulty. 

A  great  variety  of  foods,  such  as  meat,  fruit,  eggs,  and  even  milk, 
can  be  successfully  kept  by  drying.  According  to  Rohn's  classifica- 
tion the  following  groups  of  foods  can  be  kept  by  this  method : 

Group      I  .      .      .   ;-.      .      .  Protein  Foods 

Group    II  .      .      .      .      .  Carbohydrate  Foods 

Group  III  .....  Proteins     +     Carbohydrates 

Group  IV Acids     +     Proteins     +     Carbohydrates 

Pressure.— The  use  of  pressure  for  the  preservation  of  foods 
is  yet  in  the  experimental  stage.  Hite  and  coworkers  found  that  the 
bacteria  which  cause  spoilage  in  many  fruits  can  be  killed  by  press- 
ure. Apple  juice  kept  for  five  years  after  being  subjected  to  a 
pressure  of  from  90,000  to  120,000  pounds.  Peaches  and  pears 
exposed  to  a  pressure  of  60,000  pounds  for  thirty  minutes  never 


METHODS  OF  PRESERVING  FOODS  407 

spoiled.  Inconsistent  'results  were  obtained  with  blackberries, 
raspberries,  and  tomatoes,  thus  indicating  that  more  work  is  neces- 
sary before  it  can  be  used  on  a  commercial  scale.  Larson,  Hartzell 
and  Diehl  found  that  a  direct  pressure  of  6000  atmospheres  kills 
non-spore-forming  bacteria  in  fourteen  hours.  A  pressure  of 
12,000  atmospheres  for  the  same  length  of  time  is  required  to  kill 
spores.  They  think  sterilization  by  means  of  pressure  may  prove 
valuable  from  a  medical  viewpoint  as  cultures  so  killed  were  found 
very  effective  in  immunization.  They  are  disposed  to  attribute  the 
sterilization  to  the  sudden  change  in  the  osmotic  tension  of  the  fluid 
in  which  the  bacteria  were  suspended.  However,  Bridgman's 
results  indicate  that  it  may  be  due  to  the  coagulation  of  the  bacterial 
protoplasm. 

Canning.—- This  process  in  most  cases  leaves  the  food  sterile. 
It  is,  therefore,  a  sanitary  safeguard,  and  can  be  used  with  most 
meats,  fruits,  and  vegetables,  and  if  properly  conducted  yields  very 
satisfactory  products. 

The  method  used  and  the  success  met  with  varies  with  the 
different  products  and  their  condition  at  the  time  of  canning.  Acid 
foods  or  those  containing  large  quantities  of  soluble  constituents 
are  canned  with  considerable  ease  as  compared  with  the  neutral 
substances  (corn,  peas,  and  beans). 

The  various  methods  used  may  be  arranged  under  three  groups: 

1.  The  heating  of  the  products  under  pressure  for  a  sufficient 
time  to  sterilize.    This  method  although  it  requires  the  use  of  an 
autoclave  is  more  efficient  and  requires  less  time  than  the  other 
methods.     It  is  used  very  extensively  in  large  canneries,  whereas 
the  intermittent  and  continuous  methods  are  used  to  a  greater  extent 
in  the  home. 

2.  The  intermittent  method  consists  of  heating  the  products 
on  three  successive  days,  maintaining  the  food  at  a  temperature 
between  heating  such  that  spores  will  vegetate.     The  objection  to 
this  method  is  the  time  necessary  in  the  preparation  of  the  finished 
product,  and  anaerobic  organisms  may  not  germinate  in  the  intervals 
between  heating  but  may  later  with  the  production  of  toxins. 

3.  The  continuous  or  cold-pack  method  is    being    extensively 
used  of  late,  but  Dickson  and  later  Thorn  and  coworkers  have  shown 
that  the  temperature  is  not  always  sufficient  to  insure  the  death  of 
all  injurious  organisms. 

Sugar  and  $aft.— Sugar  and  salt  preserve  by  increasing  osmotic 
pressure  and  are  very  extensively  used  as  they  are  without  injury 
upon  the  health  of  the  consumer. 

Sugar  is  largely  used  in  the  manufacture  of  jellies  and  preserves. 
These  substances  are  cooked  in  the  preparation,  and  this  together 
with  the  high  osmotic  pressure  of  the  solution  renders  them  free  from 
pathogens. 


408  PRESERVATION  OF  FOOD 

Salt  is  extensively  used  in  the  preservation  of  meats  and  pickles, 
and  our  knowledge  concerning  the  action  of  salt  is  more  exact  than 
it  is  concerning  sugar. 

Tanner  lists  the  various  reasons  which  have  been  ascribed  for  the 
keeping  powers  of  salt  as  f ollowrs : 

I.  Exerts  a  poisonous  action. 

II.  Renders  the  moisture  unavailable  for  microorganisms. 

III.  Destroys  the  cells  by  plasmolysis. 

Salt  does  not  render  the  medium  sterile  but  exerts  a  selective 
action  upon  the  bacterial  flora.  A  -7  to  10  per  cent,  solution  of  salt, 
according  to  Stadler,  inhibited  the  growth  of  the  following  organisms : 
B.  Coli  commune,  B.  morbificdus  boms,  B.  enteriditis,  B.  (proteus) 
vulgaris,  and  B.  botulinus. 

De  Freytag  and  Stadler  found  that  a  saturated  salt  solution  had 
the  following  effect  upon  bacteria : 

INFLUENCE   OF  CONCENTRATED   SALT   SOLUTION   ON   BACTERIA. 

Author.  Organism.  Observation. 

Freytag       .      .      .  B.  anthracis  Not  killed  after  a  number  of  hours. 

Freytag  B.  anthracis  spores     Not  killed  in  six  months. 

Freytag       .      .      .  B.  typhosus  Killed  after  five  months. 

Stadler  ....  B.  typhosus  Not  killed  in  six  weeks. 

Freytag       .      .      .  B.  diphtherice    .  Not  killed  in  three  weeks. 

Stadler  ....  B.  diphtherice  Not  killed  in  four  and  a  half  weeks. 

Freytag  B.  tuberculosis  Not  killed  in  three  months. 

Stadler  ....  B.  pestis  Not  killed  in  sixteen  weeks. 

Homer  found  that  B.  botulinus  does  not  develop  in  media  con- 
taining over  6  per  cent,  of  salt,  and  he  considers  meat  which  is 
properly  covered  with  brine  safe.  But  much  higher  concentrations 
—12  to  19  per  cent,  acting  for  seventy-five  days— are  necessary  to 
destroy  the  bacteria,  and  even  then  ptomains  which  had  previously 
been  formed  in  the  food  would  not  be  rendered  harmless. 

It  is  quite  evident  from  these  results  that  salt  is  an  efficient  food 
preservative.  It  does  not,  however,  destroy  pathogens,  and  in 
dilute  solutions  the  organisms  involved  in  food-poisoning  may 
develop. 

Chemical  Preservatives.— All  authorities  are  agreed  that  the 
preservation  of  food  by  drying,  refrigeration,  heating,  canning, 
salting,  and  preserving  with  sugar  is  justified  on  theoretical  grounds 
as  well  as  practical  experience,  whereas  the  use  of  sulphites  in  sausage 
and  chopped  meat,  the  addition  of  formaldehyd  to  milk,  or  of  boric 
acid  or  sodium  flourid  to  butter  are  objectionable  from  the  stand- 
point of  public  health .  The  addition  of  sulphites  to  meat  is  especially 
objectionable,  as  it  places  in  the  hands  of  the  unscrupulous  dealer  a 
method  of  concealing  the  signs  of  decomposition  in  meat,  in  addition 
to  being  injurious  to  the  health. 

The  use  of  other  preservatives  such  as  benzoic  acid  and  sodium 


CHEMICAL  PRESERVATIVES  409 

benzoate  is  defended  by  some  authors,  while  others  argue  that  any 
chemical  which  is  poisonous  in  large  quantities  should  be  considered 
as  poisonous  in  small  quantities  until  the  contrary  is  proved.  This 
can  be  determined  only  by  tests  extending  over  long  periods,  for 
whereas  one  dose  may  not  be  injurious  the  continuous  use  may. 
So  it  is  best  to  exclude  as  far  as  practical  the  use  of  chemical  pre- 
servatives from  food.  The  subject  is  well  summarized  by  Jordan 
as  follows: 

"The  remedy  is  obvious  and  has  been  frequently  suggested— 
namely,  laws  prohibiting  the  addition  of  any  chemical  to  food  except 
in  certain  definitely  specified  cases.  The  presumption  then  would 
be— as  in  truth  it  is— that  such  chemicals  are  more  or  less  dangerous, 
and  proof  of  innocuousness  must  be  brought  forward  before  any  one 
substance  can  be  listed  as  an  exception  to  the  general  rule.  Such 
laws  would  include  not  only  the  use  of  chemicals  or  preservatives, 
but  the  employment  of  substances  to  '  improve  the  appearance'  of 
foodstuffs.  As  already  pointed  out,  the  childish  practice  of  arti- 
ficially coloring  foods  involves  waste  and  sometimes  danger.  It 
rests  on  no  deep-seated  'human  need;  food  that  is  natural  and 
un tampered  with  may  be  made  the  fashion  just  as  easily  as  the  color 
and  cut  of  clothing  are  altered  by  the  fashionmonger.  The  incor- 
poration of  any  chemical  substance  into  food  for  preservative  or 
cosmetic  purposes  could  wisely  be  subject  to  a  general  prohibition, 
and  the  necessary  list  of  exceptions  (substances  such  as  sugar  and 
salt)  should  be  passed  on  by  a  national  board  of  experts  or  by  some 
authoritative  organization  like  the  American  Public  Health  Associa- 
tion." 

An  advance  in  the  right  direction  was  made  by  the  passage  of  the 
National  Food  and  Drug  Law  in  1906.  This  is  being  rapidly 
incorporated  in  the  statutes  of  the  various  states.  According  to 
this  law  a  food  is  adulterated : 

1 .  "If  any  substance  has  been  mixed  and  packed  with  it  so  as  to 
reduce  or  lower  or  injuriously  affect  its  quality  or  strength. 

2.  "If  any  substance  has  been  substituted  wholly  or  in  part  for 
the  article. 

3.  "If  any  valuable  constituent  of  the  article  has  been  wholly  or 
in  part  abstracted. 

4.  "If  it  is*  mixed,  colored,  powdered,  coated,  or  stained  in  any 
manner  whereby  damage  or  inferiority  is  concealed. 

5.  "If  it  contains  any  poisonous  or  other  added  deleterious 
ingredient  which  may  render  such  article  injurious  to  health. 

6.  "If  it  consists  in  whole  or  in  part  of  a  filthy,  decomposed  or 
putrid  animal  or  vegetable  substance  or  any  portion  of  an  animal 
unfit  for  food,  whether  manufactured  or  not,  or  if  it  is  the  product 
of  a  diseased  animal  or  one  that  has  died  otherwise  than  by 
slaughter." 


410  PRESERVATION  Of1  FOOD 

Those  chemical  preservatives  concerning  which  there  is  a  question 
as  to  their  influence  upon  the  health  need  only  be  listed  on  the  article 
leaving  the  consumer  to  decide  for  himself  as  to  whether  he  cares  to 
use  it. 

REFERENCES. 

Rosenau:  Preventative  Medicine  and  Hygiene. 
Tanner:  Bacteriology  and  Mycology  of  Foods. 
Wiley:  Foods  and  Their  Adulteration. 


CHAPTER  XXXVI. 
BACTERIA  IN  THE  ARTS  AND  INDUSTRIES. 

BACTERIA  play  an  ever-increasing  part  in  the  arts  and  industries 
and  man  is  learning  that  the  majority  of  them  are  his  friends  and  not 
his  enemies.  In  addition  to  the  processes  considered  in  the  preced- 
ing pages  bacteria  play  a  leading  role  in  many  important  industries, 
a  few  of  which  are  briefly  considered  below. 

Alcoholic  Fermentation. —The  development  of  bacteriology  as  a 
science  is  intimately  associated  with  the  history  of  fermentation. 
Some  of  Pasteur's  classic  studies  dealt  with  this  subject  and  ever 
since  it  has  commanded  considerable  attention. 

Although  from  a  commercial  viewpoint  the  yeasts  are  of  first 
importance  in  alcoholic  fermentation,  yet  there  are  many  bacteria 
which  produce  alcohol,  for  instance : 

B.fitzianus,  ferments  glycerin  with  the  formation  of  ethyl  alcohol. 

B.  ethaceticus  ferments  glycerin,  starch,  sucrose,  lactose,  glucose, 
mannite,  and  arabinose  with  the  formation  of  ethyl  alcohol  and 
acetic  acid. 

A  number  of  bacteria,  chief  among  which  are  B.  butylicus,  B. 
B.  orthobutylicus,  B.  amylozyme,  and  Beijerinck's  genus  Granulo- 
bacter,  ferment  carbohydrates  with  the  production  of  butyric  acid. 

Recently  Northrop  and  coworkers  have  outlined  a  method  of 
producing  acetone  on  a  commercial  scale,  ethyl  alcohol  being  a  by- 
product. The  organism  used  is  B.  aceto-ethylicum  which  acts  on  a 
solution  of  beet  molasses.  The  fermentation  yielded  from  8  to  8.5 
per  cent,  of  the  sugar  as  acetone  and  20  to  21  per  cent,  as  ethyl 
alcohol. 

Milk  usually  undergoes  lactic  acid  fermentation,  yet  Koumiss, 
Matzoon,  Keffir,  and  Leben  all  contain  alcohol  and  bacteria  play  an 
important  role  in  their  fermentation. 

Vinegar.— Many  species  of  bacteria  have  been  described  which 
produce  acetic  acid.  They  are  all  closely  related  but  differ  slightly 
in  morphology  and  fermentative  power.  It  is  believed  that  the 
oxidation  of  the  alcohol  is  due  to  an  intracellular  enzyme.  All  of 
the  organisms  are  bacilli  and  a  few  of  the  most  common  species 
are  as  follows: 

Bacterium  pasteurianum — non-motile  rods,  1/z  x  2^,  that  do  not 
form  spores.  Their  optimum  temperature  is  about  34°  C.  They 
develop  best  in  solutions  not  over  9.5  per  cent,  of  alcohol  and 
produce  under  favorable  conditions  about  6  per  cent,  of  acetic  acid. 


412  BACTERIA  IN  THE  ARTS  AND  INDUSTRIES 

Bacterium  schiitzenbachi  and  several  related  species  are  the  main 
factors  in  the  production  of  vinegar  by  the  quick-vinegar  process. 
The  organisms  vary  considerably  in  size— 0.3 — 0.4/z  x  3 — 6ju.  Their 
optimum  temperature  is  25° — 30°  C.  They  produce  as  high  as 
11.5  per  cent,  acetic  acid.  In  the  absence  of  sufficient  alcohol  the 
acetic  acid  may  be  oxidized  by  them  to  carbon  dioxid  and  water. 

Two  methods  of  preparing  vinegar  are  in  general  use— (1)  the 
Orleans  and  (2)  the  Quick,  or  German  Method. 

1.  The  Orleans  Method  is  the  oldest  commercial  method  and 
produces  vinegar  of  the  highest  quality.     There  are  many  modifica- 
tions of  this  method  but  they  all  contain  essentially  the  same 
principles.    The  filtered  wine  is  placed  in  barrels  or  covered  vats 
furnished  with  openings  so  the  entrance  of  air  is  facilitated  and  can 
be  controlled.    The  receptacle  is  filled  about  two-thirds  full  of  a 
mixture  of  four  parts  of  good,  new  vinegar  and  six  parts  of  wine, 
preferably  that  which  has  been  pasteurized  at  55°  C.     At  times 
there  is  placed  a  light  wooden  grating  which  floats  and  helps  to 
support  the  bacterial  film.     A  small  quantity  of  a  good  bacterial 
film  is  placed  in  as  a  starter.     Periodically  a  portion  of  the  contents 
is  drawn  off  and  replaced  by  wine,  and  so  the  process  continues. 

2.  In  the  Quick  or  German  Method  the  liquid  to  be  acetified 
is  allowed  to  trickle  through  barrels  filled  with  beech  chips,  the 
pressed  pomace  of  red  wines,  rattan  shavings,  corn  cobs,  or  charcoal. 
Although  the  main  function  of  these  is  to  increase  aeration,  yet  the 
best  results  are  obtained  with  the  beech  shavings  or  pomace. 

Sauerkraut.— The  cabbage  is  cleaned,  cut  into  pieces  of  con- 
venient size,  and  tightly  packed  with  from  1  to  3  per  cent,  of  salt 
into  wooden  or  earthen  vessels  on  the  top  of.  which  is  placed  a 
weighted  perforated  cover.  This,  together  with  the  osmotic  press- 
ure of  the  salt,  draws  from  the  vegetable  considerable  water.  The 
respiration  of  the  cells  of  the  leaf  and  the  yeast  soon  remove  all 
oxygen.  The  mass  undergoes  lactic  acid  fermentation  which  in 
time  reaches  from  0.5  to  1  per  cent.  The  brine  is  then  drawn  off 
and  replaced  by  4  to  8  per  cent,  salt  solution.  In  this  the  vegetable 
will  keep  for  a  considerable  time.  Many  substances  are  produced 
in  the  process,  the  chief  of  which  are  lactic  acid,  alcohol,  succinic 
acid,  volatile  acids,  mannite,  amid  bodies,  carbon  dioxid,  hydrogen, 
methane,  and  various  aromatic  esters. 

The  bacteria  responsible  for  the  process  come  from  the  vegetable. 
Although  Weiss  has  isolated  65  different  species  of  bacteria  from 
sauerkraut,  probably  the  principal  changes  are  due  to  a  few  species. 
The  lactic  acid  is  usually  produced  by  Streptococcus  lacticus  and 
Bacterium  lactus  acidi. 

By  similar  means  other  vegetables— stringbeans,  cucumbers, 
etc.— may  be  preserved. 


ENSILAGE  413 

Ensilage.— The  changes  through  which  ensilage  passes  during  its 
curing  was  looked  upon  a  few  decades  ago  as  being  entirely  microbic 
in  origin,  but  due  to  the  work  of  Babcock  and  Russell  (1906-10) 
opinion  swung  in  the  opposite  direction  to  such  an  extent  that 
microorganisms  were  generally  considered  as  of  little  if  any  signifi- 
cance in  the  normal  fermentation  of  silage.  Later  (1912)  Esten 
and  Moson  considered  the  process  entirely  bacteriological.  Three 
chief  fermentations  were  thought  to  take  place:  the  lactic  acid, 
alcoholic,  and  acetic  acid  fermentation.  The  lactic  acid  fermenta- 
tion was  thought  to  be  due  to  organisms  similar  to  those  concerned 
in  the  souring  of  milk.  It  was  also  believed  by  these  workers  that 
yeasts  cause  an  alcoholic  fermentation  and  that  acetic  acid  bacteria 
then  oxidize  the  alcohol  so  formed  to  acetic  acid.  Samarani  con- 
cludes that  the  acetic  acid  fermentation  in  silage  is  due  to  the  res- 
piration of  the  plant  cells,  while  the  lactic  acid  fermentation  is  due 
to  bacterial  action.  The  organisms  responsible  for  the  latter  proc- 
ess were  identified  by  him  as  a  bacillus  and  a  coccus  which  occurred 
in  about  equal  proportions.  The  former  he  designated  as  the  B. 
acidi  lactici  of  Hulppe,  and  the  latter  was  considered  identical  with 
the  common  streptococcus  of  milk. 

Counts  made  by  Sherman  on  silage  juice  showed  the  presence  of 
from  1,500,000,000  to  4,800,000,000  per  cubic  centimeter,  most  of 
which  were  slender  rods,  and  he  considered  the  organisms  con- 
cerned to  be  nearly  related  to  the  B.  bulgaricus  group  of  milk  and  the 
B.  acidophilus  groups. 

However,  the  temperature,  kind  of  silage,  and  other  factors 
would  govern  the  bacterial  flora,  and  Gorini  distinguishes  four  types 
of  grass-silage  prepared  in  pits,  depending  upon  the  predominating 
type  of  bacteria  as  follows:  (1)  Butyric,  (2)  lactic,  (3)  putrefactive, 
and  (4)  sterile  or  atypical.  If  the  silage  stage  reaches  a  temperature 
of  60°  C.  butyric  organisms  predominate;  if  50°  C.  lactic  organisms 
prevail;  putrefaction  occurs  at  lower  temperatures,  and  sterile  or 
atypical  when  the  mass  becomes  superheated.  Butyric  silage  is 
objectionable  because  of  the  odor  and  taste  which  it  is  apt  to  impart 
to  the  milk  and  the  bacteria  which  enter  from  the  surroundings 
render  the  milk  unsuitable  for  cheese-making. 

The  acid-produqing  bacteria  of  silage  are  found  constantly  on 
corn  fodder,  so  that  silage  made  from  corn  is  always  amply  seeded 
with  the  organisms,  but  Gorini  achieved  considerable  success  by 
inoculating  fresh  grass-silage  with  lactic  acid  bacteria,  and  Crolhois 
found  that  the  inoculating  beet  silage  with  lactic  acid  organisms 
preserves  it  better,  furnishes  a  more  nutritive  product,  and  sup- 
presses the  diseases  to  which  the  cattle  fed  on  non-inoculated  pulp 
are  subject. 

At  least  from  a  theoretical  basis  this  would  seem  quite  probable, 
for  it  is  known  that  beets  contain  in  addition  to  many  other  products 


414  BACTERIA  IN  THE  ARTS  AND  INDUSTRIES 

cholin  and  betain.  The  quantity  of  this  last  product  in  the  ripe 
beet  is  0.1  per  cent.,  in  the  unripe  beet  0.25  per  cent.,  and  in  the  beet 
molasses  as  high  as  3  per  cent.  The  quantity  may  be  even  greater 
in  the  leaves  and  upper  part  of  the  beet  than  in  the  main  beet. 

Now  certain  organic  reactions  are  known  which  relate  these 
products  to  a  toxic  substance.  Cholin  on  oxidation  and  the  subse- 
quent elimination  of  a  molecule  of  water  passes  into  betain : 

CH>CH2OH  CH2 

/  /     \ 

(CH3)3N  ,       +     20     =     (CH3)3N  C=0     +     2H2O 

OH  ^  O 

Cholin.  Betain. 

This  is  a  typical  oxidation  and  dehydration  reaction  which  could 
be  brought  about  by  mold  or  bacteria  under  aerobic  conditions, 
whereas  betain  can  be  converted  into  muscarin  through  being  made 
to  take  up  water  and  reduced  thus: 

CH2  CH2— CHO.H2O 

/      \  / 

(CH3)3N  C=O     +    H2     +    H2O  =  (CH3)3N 

^  O  y  OH 

Betain.  Muscarin. 

This  is  a  reaction  which  theoretically  could  be  catalyzed  by  bac- 
teria or  molds  and  would  probably  occur  under  anaerobic  conditions. 

It,  therefore,  appears  plausible  that  under  appropriate  tempera- 
ture, moisture,  aeration,  and  microflora  there  may  develop  in  beet 
silage  toxic  compounds. 

Retting.— The  separation  of  the  fibers  of  flax  and  hemp  is  brought 
about  by  a  complex  fermentation  in  which  bacteria  dissolve  certain 
pectin  bodies  which  cement  the  fibers  together.  The  reaction 
occurs  best  at  a  temperature  of  30°  to  32°  C.  and  is  due  to  many 
species  of  bacteria.  In  the  water-retting  of  hemp,  the  anaerobic 
butyric  acid  bacteria  (Clostridia)  play  a  leading  role,  and  the  water- 
retting  of  flax  is  ascribed  to  a  specific  anaerobic  bacillus  ( Granu- 
lobacter  pectinovorwri) . 

Tanning.— Animal  skins  are  tanned  in  order  to  increase  their 
resistance  to  decomposition  and  also  to  increase  their  adaptability 
to  the  various  purposes  to  which  leather  is  put.  In  tanning  bacteria 
play  important  parts.  When  the  skin  is  soaked  in  baths  rich  in 
organic  matter  an  energetic  bacterial  flora  soon  develops  which 
quickly  softens  the  hide.  Bacteria  cause  the  depilation  and  removal 
of  the  hair  by  which  the  dermis  is  separated  from  the  epidermis  and 
the  hair  which  accompanies  it.  This  is  true  in  the  sweating  and  lime 
methods,  whereas  the  alkaline  sulphid  and  arsenic  sulphid  are  both 
chemical  methods. 

The  third  step  in  the  process,  is  conducted  in  the  excrement  or 


VACCINES  415 

bathing  tubs  which  contain  the  droppings  of  hens,  pigeons,  and  dogs. 
Here  a  true  digestion  of  the  hide  is  carried  on  by  bacteria.  Wood 
has  isolated  90  species  of  bacteria  from  such  a  tube,  no  one  of  which 
possessed  the  power  of  bringing  about  the  desired  change,  but  all 
acting  conjointly  gave  the  desired  product.  The  heated  hides  are 
next  placed  in  a  tan  pit  or  in  bark  liquor.  Numerous  bacteria 
yeast  and  molds  occur  in  the  bark  liquor  and  play  a  part  in  the 
finishing  of  the  product. 

Vaccines.— A  vaccine  is  a  killed  or  weakened  (attenuated)  sus- 
pension of  organisms  to  be  inoculated  into  the  body  for  the  purpose 
of  causing  the  development  of  an  active  immunity. 

The  vaccine  is  usually  prepared  from  a  fresh  twenty-four-hour 
growth  of  the  microorganism  on  agar.  The  surface  growth  only  is 
taken,  thus  avoiding  secondary  metabolic  products  which  may  be 
formed.  The  cultures  are  usually  killed  by  exposure  to  heat  at 
from  53°  to  60°  C.  for  one  hour.  High  heat,  while  certain  to  kill 
the  virus,  is  undesirable  for  the  reason  that  it  coagulates  the  protein 
substances  in  the  bacterial  cell  and  otherwise  alters  its  chemical 
structure.  The  closer  the  vaccine  approaches  the  virus  the  better 
the  result  and  higher  the  resulting  immunity.  For  this  reason  many 
workers  prefer  to  kill  the  bacteria  with  chemicals,  carbolic  acid, 
chloroform,  or  some  other  suitable  germicide. 

The  attenuated  virus  is  obtained  by  passing  the  microorganism 
through  the  body  of  some  animal,  as  smallpox  through  the  heifer 
by  which  its  virulence  for  man  is  reduced.  At  other  times  it  is 
grown  under  adverse  conditions— high  temperatures,  artificial  media, 
or  in  the  presence  of  antiseptics,  after  which  it  becomes  less  virulent. 
Drying  is  used  in  the  case  of  the  virus  of  rabies. 

In  the  preparation  of  antitoxins  the  bacterial  cell  or  some  of  its 
products  are  injected  into  a  suitable  animal,  and  after  sufficient 
time  has  elapsed  blood  is  drawn  and  after  appropriate  treatment  is 
used  for  the  cure  or  prevention  of  disease. 

REFERENCES. 

In  addition  to  the  references  listed  at  the  end  of  the  several  chapters,  the  fol- 
lowing have  been  freely  consulted  and  to  these,  the  students  are  referred  for  further 
information.  The  date  given  is  that  in  which  the  first  volume  appeared. 

Abstracts  of  Bacteriology,  1917,  vol.  i. 

Agricultural  Index,  1916,  vol.  i. 

Biedermann's  Centralblatt  fur  Agrikulturchemie,  1872,  Bd.  i. 

Botanical  Gazette,  1876,  vol.  i. 

Centralblatt  fur  Bakteriologie,  1887,  Abt.  I,  Bd.  i. 

Centralblatt  fur  Bakteriologie,  1892,  Abt.  II,  Bd.  i. 

Chemical  Abstract,  1907,  vol.  i. 

Experiment  Station  Record,  1888,  voh  i. 

Comptes  Rendus  Academic  des  Sciences,  1835,  vol.  i. 

Experiment  Station  Bulletins. 

International  Catalogue  of  Scientific  Literature,  1911. 

Jahresbericht  iiber  die  Fortschritte  der  Agrikulturchemie »  1858,.  Bd.  i. 


416  BACTERIA  IN  THE  ARTS  AND  INDUSTRIES 

Jahresbericht  iiber  die  Landwirtschaft,  1886,  Bd.  i. 

Journal  of  Agricultural  Science,  1906,  vol.  i. 

Journal  of  the  American  Chemical  Society,  1875,  vol.  i. 

Journal  of  the  American  Medical  Association. 

Journal  of  Agricultural  Research,  1913,  vol.  i. 

Journal  of  the  Infectious  Diseases,  1904,  vol.  i. 

Journal  of  American  Society  of  Agronomy,  1910,  vol.  i. 

Journal  of  Bacteriology,  1916,  vol.  i. 

Journal  of  Biological  Chemistry,  1906,  vol.  i. 

Journal  of  Industrial  and  Engineering  Chemistry,  1909,  vol.  i. 

Journal  fur  Landwirtschaft,  1853,  Bd.  i. 

Landwirtschaftliches  Jahrbuch  der  Schweiz,  1887,  Bd.  i. 

Landwirtschaftliche  Versuchs-Stationen,  1859,  Bd.  i. 

Phytopathology,  1911,  vol.  i. 

Soil  Science,  1916,  vol.  i. 

United  States  Department  of  Agriculture  Bulletins. 


INDEX  OF  AUTHORS. 


A 


ABENHAUSEN,  388 

Adler,  180 

Allen,  141 

Allison,  262      • 

Ampola,  245 

Ampola  and  Garino,  241 

Anderson,  382 

Andre,  234 

Andrews,  392 

Armsby,  249 

Armstrong,  75 

Arrhenius,  95,  99,  106,  111 

Ascher,  388 

Ashby,  219,  221,  253,  273 

Atkinson,  299 

Atwater,  291 

Aubin,  217 

Aujeszky,  388 

Avery,  249 

Ayers,  385 


B 


BABCOCK  and  RUSSELL,  413 

Ball,  299 

Barbieri,  310 

Barlow,  299,  303 

Barthel,  153 

Bassler,  156 

Baumann,  210 

Bayliss,  73,  81 

Bazarewski,  230,  240,  245 

Beatty,  382 

Beckwith,  123 

Beddies,  226,  230 

Beijerinck,  27,  57,  93,  144,  251,  263, 

266,  268,  276,  292,  293,  297,  307,  411 
Beijerinck  and  Van  Delden,  53,  251, 

254,  275 
Beik,  382 
Benin^e,  388 
Benjamin,  307 
Bergonzini,  56 
Berman,  70 
Bernard,  65 
Berthelot,  60,  249,  250,  261,  267,  278, 

281,  287 
27 


Berzeluis,  71 

Biedermann,  415 

Bienstock,  190 

Binot,  337 

Birner,  249 

Bizzell,  131,  140,  219,  233,  234 

Black,  401 

Bodenstein,  74 

Boldnau,  403 

Bolley,  387 

Bellinger,  227,  277 

Bolton,  391 

Bomhoff,  388 

Bonazzi,  271 

Borisson,  78 

Bornemann,  153 

Bottomley,  138,  235,  236,  274,  286 

Bouilhac,  122 

Boullanger,  221,  225 

Boullanger  and  Massol,  221,  225 

Bouonami,  18 

Boussingault,  209,  218,  227,  248 

Boyd,  387 

Breal,  218,  240 

Bredemann,  157,  251,  264 

Brenchley,  122 

Brett,  390 

Briailles,  130 

Bridgman,  106,  407 

Brieger,  189 

Briggs,  201 

Briscoe,  152,  388,  389 

Briscoe  and  MacNeal,  388,  389 

Brown,  41,  96,  129,  135,  140,  141,  142, 

144,  152,  158,  162,  174,  199,  200,  203, 

233,  283 

Brown  and  Allison,  262 
Brown  and  Hitchcock,  146 
j  Bmnchorst,  291 
Brusaferro,  388 
Buchanan,  30,  94,  106 
Buchner,  72,  79,  83,  101,  343 
Buck,  387 
Buff  on,  19 
Buhlert,  292 
Bujwid,  382 
Burgess,  122,  124,  140,  195,  252,  255, 

276 

Burri,  216,  226,  240 
Burri  and  Stutzer,  216,  226,  240 


418 


INDEX  OF  AUTHORS 


Burrill  27,  295,  298,  300,  302,  318 
Burrill  and  Hansen,  295,  298,  299,  300, 

318 

Bushnell  and  Mauer,  392 
Busley,  224 
Bychiklin,  175,  232 
Bychiklin  and  Skalski,  232 


CALDWBLL,  392,  393 

Campbell,  382 

Carbone,  202 

Carey,  394 

Caron,  151,  241,  242,  250,  284,  285 

Carter,  159,  180,  199,  201 

Causemann,  157 

Ceffi-Zuco  and  Heraeus,  210 

Chamberlain,  182 

Chapin,  352,  399 

Chester,  51,   141,   161,   163,   164,   196, 

217,  232,  238,  251 
Chestnut,  395 
Chick,  111,  113,  117 
Chick  and  Martin,  113 
Chisholm,  359 
Christen,  98 

Christensen,  254,  258,  259 
Christensen  and  Larson,  254 
Cienkowski,  55 
Clark  and  Gage,  123 
Clausen,  95 
Clayton,  386 
Coggi,  388 

Cohn,  21,  46,  55,  57,  190 
Coleman,  136,  138,  221,  222,  227,  256 
Colton,  389 
Conn,  64,  96,  162,  164,  165,  166,  167, 

168,  170,  197,  372,  376 
Conn  and  Brown,  96 
Cook,  182 
Coudon,  194 
Cramer,  61 
Crochetelle,  218,  220 
Crolhois,  413 
Crooks,  183 
Gumming,  391 
Cunningham,  126,  128,  173 
Cunningham  and  Lohnis,  126,  281 
Czapek,  118 


DAFERT  and  BOLLINGER,  227,  277 
Dakin,  113,  114,  115,  117,  349 
Dakin  and  Dunham,  113,  117 
Dallar,  382 

Darbishire  and  Russell,  130 
Davenport,  297 
Davis,  252 
Davy,  239 


Dawson,  303 
De  Barv,  165 
Deherain,  145,  151,  227,  230,  231,  232, 

235,  243,  249,  274,  328,  330 
Deherain  and  Derm -ussy,  151,  227 
Delepine,  382 
Delwiche,  156 
Demolon,  123 

Demoussy,  151,  22^,  227,  231 
Depetit,  240,  244 
DeSchweinitz  and  Dorset,  59 
Dezani,  142 

Dickson,  400,  401,  403,  407 
Diehl,  407 

Doleris  and  Pasteur,  190 
Don,  359 
Done,  390 
Donk,  394 
Dorset,  59 
Douglass,  386 
Drouin,  307 

DuBois  and  Raymond,  105 
Duclaux,  98,  241 
Duggar  and  Davis,  252 
Dumas,  208 
Dumont,  145,  218,  220 
Dumont  and  Crochetelle,  218,  220 
Duncan,  181 
Dunham,  113,  117 
Dusch,  20 

Duschechkin,  153,  158 
Dvarak,  263 
Dzierzbicki,  199,  202,  203,  258 


E 


EBER,  382,  388 

Ebermayer,  141 

Eberth,  352 

Effront,  190,  193,  206 

Egorou,  128 

Ehrenberg,  54,  56,  123,  141 

Ehrlich,  84 

Einecke,  152 

Ellis,  180 

Elwell,  218 

Emerson,  252 

Emmerich,  152 

Emmerling,  197 

Engberding,  140,  143,  144,  151,  158 

Engelmann,  109 

Erickson,  291 

Escherich,  56,  190 

Esten  and  Moson,  413 

Euler,  81 

Evans,  315 


F 


FABRICIUS  and  VON  FEILITZEN,  151 
Feilitzen  von,  151,  225 
Fernan  and  Pauli,  103 


INDEX  OF  AUTHORS 


419 


Field,  382 

Fischer,  29,  77,  9  r,  98,  140,  151,  153, 

158,  188,  200,  203,  219,  222,  254,  276 
Fletcher,  132 
Flexner,  391 

Fliigge,  38,  57,  165,  166,  168,  190 
Folwell,  367 
Ford,  396 

Forster,  96  4 

Fraenkel,  216 
Fran9ois,  118 

Frank,  53,  127,  210,  274,  291,  315 
Frankfurt  and  Duschechkin,  153,  158 
Frankland,  165,  167,  210 
Fraps,  139,  227 
Fred,  129,  131,  133,  136,  137,  244,  267, 

304,  306,  307 
Fred  and  Davenport,  297 
Fred  and  Gainey,  136 
Fred  and  Hart,  145 
Fresenius,  198 
Freudenreich,  251,  273,  377 
Freytag,  408 
Friedlander,  83 
Froehde,  239 
Fromberz,  206 
Frost,  54 

Fuhrmann,  79,  306 
Fuller,  193,  345,  360,  361,  362,  367 
Fuller  and  Johnson,  345 
Fulmer,  266,  267 
Fulmer  and  Fred,  267 


G 


GAFKY,  352 

Gage,  123,  196 

Gain,  3 12,  313 

Gainey,  136,  203,  228 

Gandechon,  225 

Garino,  241 

Garaian,  295 

Gartner,  216 

Gasperini,  169 

Gay,  355 

Gay-Lussac,  77 

Gayon,  240,  244,  328,  330 

Gayon  and  Depetit,  240,  244 

Gerlack  and  Vogel,  69,  154,  251,  257, 

307 

Gibbs,  212,  213,  238 
Gilbert,  191,  209,  235,  240,  290 
Giltner  and  Langworth,  276 
Girard,  !rt7 
Goc  .3wski,  221 
Golding,  308 
Goler,382 
Goodsir,  55 
Gorini,  413 
Goss,  390 
Gottheil,  165,  166 
Grassberger,  388 


Grazia,  151,  175 

Greaves,  126,  146,  149,  159,  162,  164, 

180,  199,  201,  218,  232,  234,  237,  238, 

244,  255,  277,  289,  326 
Greaves  and  Carter,  159,  180,  199,  201 
Greaves,  Stewart  and  Hirst,  238 
Green,  200,  203,  234,  260,  266,  282 
Gregory,  28 
Greig-Smith,  138,  235,  236,  297,  299, 

306,  312 

Greig-Smith  and  Bottomley,  235,  236 
Groenewege,  252 
Groning,  388 
Grove,  74 
Grunner,  118 
Guatier  and  Drouin,  307 
Guffroy,  143 
Guignard,  249 


HAAGLAND,  393 

Haas,  99 

Hadley  and  Caldwell,  392,  393 

Haeckel,  29 

Hall,  172,  288,  289 

Hamilton,  391 

Hammer,  265,  390 

Hammer  and  Goss,  390 

Hansen,  27,  295,  298,  299,  300,  318 

Hanzawa,  266 

Harden,  83,  86 

Harding,  164,  373,  389 

Harding  and  Wilson,  373 

Harracks,  359 

Harrison  and  Barlow,  299,  303 

Hart,  145 

Hartleb,  216,  217 

Hartwell,  141 

Hartz,  51,  169 

Hartzell,  407 

Hastings,  376 

Hauser,  56,  190 

Hazen,  353 

Headden,  118,  284 

Hecker,  151 

Heim,  387 

Heineman,  386 

Heinze,   128,   135,  153,  157,  231,  234, 

256,  274,  278,  282,  307 
Heller,  395 

Hellriegel,  27,  248,  274,  291,  293,  314 
Hellriegel  and  Wilfarth,  248,  291 
Hellstrom,  152,  388 
Helmholtz,  21 
Heraeus,  210 
Herbert,  328,  329,  388 
Herr  and  Berninde,  388 
Herzfield  and  Lange,  118 
Herzog,  41 
Hess,  382 
Hilgard,  141,  191,  201,  230,  235,  284 


420 


INDEX  OF  AUTHORS 


Hill;  44,  80,  157,  355,  372,  379 

Hill  and  Slack,  372 

Hills,  256,  257,  271 

Hiltner,  232,  282,  305,  307,  318 

Hiltner  and  Stormer,    129,    133,    151, 

160,  164,  257,  297 
Himesch,  388 
Hinds,  344 
Hinterberger,  197 
Hippocrates,  351 
Hirschler,  188 
Hirst,  238,  326 
Hiss  and  Zinsser,  99 
Hitchcock,  146 
Kite,  406 
Hoelling,  52 

Hoffmann  and  Hammer,  265 
Homer,  408 
Hope,  382 
Hopkins,  63,  183,  237,  276,  288,  311, 

322,326,370 

Hopkins  and  Whiting,  175 
Hoppe-Seyler,  263,  328,  329 
Hormon  and  Morgenroth,  388 
Houston,  344 
Hull  and  Rettger,  371 
Hulme,  244 
Hulppe,  413 
Hutchinson,   121,   123,   130,   135,   137, 

138,  230,  231,  235,  236,  253 


IMMENDORFF,  334 
Itano,  204 


JACKSON,  123 

Jaeger,  382,  388 

Janowski,  342 

Jeannert,  188 

Jenner,  24 

Jennings,  108 

Jensen,  52,  53,  67,  234,  242 

Jodidi,  206 

Johnson,  132,  138,  239,  345,  361,  385 

Jones,  281 

Jordan,  46,  82,  343,  344,  346,  364,  395, 

397,  403,  409 
Joshi,  226 


KAENSCHE,  397 

Kalantarov,  313 

Karg  and  Schmori,  42 

Kaserer,  52,  216,  225,  259,  260,  334 

Kayser,  194,  365,  392 

Keith,  350 


Kellermann  and  Beckwith,  123 

Kellermann  and  McBeth,  333 

Kellermann  and  Robinson,  140,  143 

Kelley,  141,  176,  199 

Kellner,  249 

Kellogg,  141 

Kendall,  37,  44,  62,  70,  88,  94,  188 

Kern,  57 

Kiesow,  249 

King,  151,  230,  231,  232,  234 

King  and  Whitson,  232,  234 

Klebs,  59 

Klein,  382 

Klimmer  and  Kruger,  295 

Knisely,  309,  310 

Knoop,  269 

Kober,  354 

Koch,  24,  54,  99,  127,  131,  133,  157, 

160,  251,  264,  265,  279,  281,  288, 

348,  352,  354,  380 
Koch  and  Seydel,  264,  265 
Kochenavsk,  219 
Kolle  and  Zetnow,  41 
Kopeloff  and  Coleman,  136,  138 
Korn,  388 

Kossowiez,  180,  187,  238 
Kossowitsch,  274 

Krainsky,  255,  264,  266,  273,  277,  287 
Kresslig,  59 
Krober,  175 
Krogh,  182 
Kronig,  112,  249 
Kruger,  140,  245,  274,  295 
Kruger  and  Heinze,  128 
Krumwiede  and  Noble,  387 
Kruse,  62,  70 

Krzemieniewski,  255,  260,  265,  268 
Kuhlmann,  239 
Kuhlmann  and  Dumas,  208 
Kuhne,  72,  215 
Kunkel,  118 
Kunnemann,  240 
Kurth,  56 


LABAVIUS,  71 

Ladd,  218 

Lafar,  94,  178,  179,  180,  187,  207,  223, 

225,  238,  318,  335 
Landolt,  210 
Lange,  118 
Langworth,  276 
Larson,  254,  407 
Latham,  123 

Laurent,  158,  241,  274,  292 
Laws  and  Gilbert,  191,  235,  240,  290 
Lazear,  27 
Leach,  62 

Leeuwenhoek,  17,  18,  42 
Lemmermann,  141,  162,  198,  244 
Lemmermann  and  Ernecke,  152 


INDEX  OF  AUTHORS 


421 


Lemmermann  and  Fresenius,  198 

Leoncini,  144 

Lery  and  Kayser,  365 

Libby,  28 

Liebig,  21,  23,  71,  189,  290 

Lipman,  C.  B.,  142,  143,  145,  146,  164, 
196,  199,  200,  218,  235,  252,  255,  260, 
277,  278,  287,  317 

Lipman,  J.  G.,  27,  60,  135,  138,  140, 
141,  152,  159,  191,  193,  197,  202,  203, 
204,  207,  238,  239,  246,  247,  251,  256, 
264,  267,  269,  288,  289,  314,  318,  334 

Lipman  and  Brown,  129,  140,  199,  200 

Lipman,  Brown  and  Owen,  140 

Lipman  and  Burgess,  122,  124,  140, 
143,  195,  252,  255,  276 

Lipman  and  Green,  203 

Lipman  and  Sharp,  146,  277 

Lipman  and  Waynick,  200,  283 

Lipman  and  Wilson,  123 

Lister,  26 

Locy,  28 

Loeb,  108,  117 

Loew,  386 

Lohnis,  38,  126,  138,  160,  161,  198,  207, 
221,  224,  238,  251,  252,  260,  262,  266, 
272,  279,  281,  282,  289,  318 

Lohnis  and  Green,  260,  266 

Lohnis  and  Smith,  272 

Lohnis  and  Pillai,  262 

Lorenz,  388 

Lumia,  145 

Lyon  and  Buzzell,  131,  140,  219,  233, 
234 


M 


MAASSEN  and  MULLER,  293 

Macaulay,  24 

Macfayden,  382 

MacNeal,  64,  388,  389 

MacNutt,  353,  369,  386 

Makrinov,  143 

Malpighi,  291 

Marchal,  194,  195,  196,  203,  204 

Marchiotti,  388 

Marcille,  221,  235 

Marconi,  382 

Markl,  388 

Marshall,  70,  94,  367 

Martelly,  190 

Martin,  113 

Mason,  359 

Massol,  221,  225 

Mauer,  392 

Mayer,  345 

McBeth,  145,  228,  231,  263,  333,  335 

McBeth  and  Smith,  228,  231 

McBeth  and  Wright,  145 

McClendon,  117 

McCoy,  375 

McLean  and  Wilson,  197,  204 

McWeeney,  398 


Mendel,  370 

Metchnikoff,  32,  370 

Meyer,  44,  59,  231 

Michel,  351 

Migula,  38,  46,  168 

Millard,  153 

Mills,  347,  353 

Miquel,  96,  336,  337,  342 

Mitscherlich,  328 

Miyaka,  204 

Moak,  371 

Mockeridge,  255,  258,  264 

Mohler,  382,  389 

Molisch,  180 

Moll,  153,  234,  282 

Montanan,  144 

Moore,  73,  251,  318 

Morgan,  131 

Morgenroth,  388 

Morrey,  18 

Moson,  413 

Mueller,  54 

Mulder,  208 

Mulford,  224,  225 

Muller,  209,  293,  382 

Munter,  170,  231 

Mlinter  and  Robson,  228 

Miintz,  157,  209,  210,  217,  222 

Miintz  and  Aubin,  217 

Miintz  and  Coudon,  194 

Murray,  281 

Myer,  196 


N 


NADSON,  173,  196 

Nathan,  70 

Navy,  400 

Neale,  156 

Needham,  19 

Neish,  232 

Nencki,  188 

Neubauer  and  Fromberz,  206 

Newton,  393 

Niklewski,  52,  153,  222 

Nishimwia,  60 

Nobbe,  293,  307,  318 

Nobbe  and  Hiltner,  307,  318 

Noble,  387 

Northrop,  411 


OBERLIN,  127 

Obermuller,  388 

Olaru,  144,  259 

Omelianski,    159,   224,  252,  264,  271, 

275,  330,  331,  332,  333 
Omelianski  and  Salunskov,  275 
Omelianski  and  Sswewrowa,  271 
Ophuls,  401 


422 


INDEX  OF  AUTHORS 


Orr,  374 

Osborne  and  Mendel,  370 

Ostwald,  75,  77 

Ott,  382 

Owen,  140,  201,  203,  219,  221 


PAGET,  28 

Pagnoul,  198 

Parker,  379,  382,  386 

Pasteur,  20,  21,  23,  24,  25,  26,  28,  72, 

182,  190,  209,  328,  337,  411 
Paterson,  141,  142,  143 
Paterson  and  Scott,  227 
Paul  and  Kronig,  112 
Pauli,  103,  105 
Pawlowsky,  382,  388 
Peck,  140,  144,  263 
Perotti,  175 
Peterson,  141,  282 
Peterson  and  Wollny,  141 
Petit,  151 
Petri,  382 
Pettenkofer,  182 
Pfeiffer,  57,  108,  156,  198,  242 
Pfulel,  387 
Phelps,  367 
Pichard,  142 
Pierce,  305 
Pillai,  262 
Platt,  210 
Plenge,  174 
Plummer,  231 
Popoff,  328 

Potter  and  Synder,  206 
Prazmowski,  57,  271,  292,  300,  304 
Prescott  and  Winslow,  344,  359 
Pringsheim,  252,  263,  264 
Prucha,  313  374,  389 
Pugh,  240 
Putnam,  240 


QUIROGA,  232 


RABINOWITSCH,  388 

Radot,  28 

Rahn,  80,  199 

Raymond,  105 

Redi,  19 

Reed,  232,  259,  283 

Reed  and  Williams,  232,  283 

Reincke,  353 

Reitz,  388 

Remy,  141,  151,  161,  255,  266,  268 

Remy  and  Fischer,  151 


Renault,  145,  223 

Rettger,  56,  67,  70,  204,  370,  371.  392, 

393 

Richards,  242,  267 
Richards  and  Rolfo,  242 
Richardson,  232 
Riviere  and  Bouilhac,  122 
Robertson,  81 
Robinson,  140,  143,  310 
Robson,  228 
Rogers,  389 
Rohn,  406 
Rolfs,  242 
Rosenau,  111,  353,  359,  367,  375,  378, 

383,  384,  386,  388,  394,  405 
Rosenbach,  51,  55,  190 
Rosing,  259,  260 
Rossi,  293,  297 
Roth,  388 
Rowland,  390 
Ruppel,  59 
Russell,  92,   121,    123,   130,    131,   136, 

137,  138,  231,  234,  236,  247,  286,  413 
Russell  and  Hutchinson,  121,  123,  130, 

131,  136,  137,  138,  236 


SACKETT,  270,  284 

Samtee,  367 

Sanarani,  413 

Sanfelice,  169 

Saper,  399 

Savage,  359,  373,  381,  386,  397 

Sawyer,  399 

Scharbekow,  382 

Schegehdahl,  355 

Schettenhelm,  174 

Schlosing,  145,  209,  210,  214,  227,  230, 

234,  274,  292,  329 
Schlosing  and  Laurent,  274,  292 
Schlosing  and  Mtintz,  210 
Schmori,  42 
Schneider,  152 
Schneidewind,  231,  274 
Schonbein,  208 
Schreiner,  193 
Schroeder  and  Brett,  390 
Schroeder  and  Dusch,  20 
Schroeter,  55,  174 
Schuchardt,  388 
Schuder,  355 
Schultz,  156 

Schultz  and  Borisson,  78 
Schulze,  20,  310 
Schulze  and  Barbieri,  310 
Schwann,  20 
Scott,  143,  227 
Sedgwick  and  MacNutt,  353 
Senus,  330 
Setchell,  96 
Severin,  142,  177,  178,  241,  250 


INDEX  OF  AUTHORS 


423 


Seydel,  264,  265 

Sharp,  146,  201,  277 

Shedd,  123 

Sherman,  413 

Shroeder,  382,  389 

Shutt,  234,  316 

Silberberg,  123 

Simon,  295 

Sirker,  135 

Skalski,  232 

Skinner  and  Sullivan,  143 

Slack,  372 

Smillie,  391 

Smirnov,  222 

Smit,  382 

Smith,  138,  228,  231,  235,  236,  239,  272, 
297,  299,  306,  310,  312,  337,  405 

Smith  and  Robinson,  310 

Snow,  351 

Snyder,  191 

Solmgen,  334 

Sorenson,  204 

Spallanzani,  19 

Sswewrowa,  271 

Stadler,  408 

Stefan,  308 

Stevens,  153 

Stevens  and  Withers,  158 

Stewart,  232,  234,  238,  326 

Stewart  and  Greaves,  232,  234 

Stigell,  151 

Stocking,  374 

Stoklasa,  60,  122,  131,  135,  152,  177, 
241,  242,  243,  244,  256,  258,  262,  268, 
274,  285,  306,  308,  309,  311 

Stoklasa  and  Vitek,  242,  243,  244 

Stormer,  129,  133,  151,  160,  257,  297 

Stranak,  256,  262 

Straus,  384 

Stutzer,  216,  217,  226,  240,  242,  309 

Stutzer  and  Hartleb,  216,  217 

Sullivan,  143 

Sulunskov,  275 

Synder,  206 


TANKER,  385,  392,  394,  403,  408,  410 

Tappeiner  and  Hoppe-Seyler,  328 

Taylor,  94,  204 

Teichert,  388 

Temple,  152,  312 

Thaysen,  138 

Thorn,  407 

Thomasen,  217 

Thomson,  75 

Thresh,  359 

Thu,  388 

Tissier  and  Martelly,  190 

Tobler,  388 

Tonney,  382 

Traaen,  277 


Trecol  and  Fremy,  23 

Treciil,  328,  332 

Trevisan,  54,  57 

Troop,  151 

Truog,  178 

Tsiklinsky,  96 

Tyndall,  23,  24,  26,  72,  337 


VAN  DELDEN,  53,  178,  251,  254,  275 

Van  Ermengen,  400,  401 

Van  Helmont,  18,  71 

Van't  Hoff  and  Arrhenius,  95,  99,  106 

Van  Tieghem,  55,  252 

Vaughan,  62,  91 

Vaughan  and  Novy,  400 

Veillon  and  Zuber,  190 

Vitek,  242,  243,  244 

Voelcker,  123 

Vogel,  69,  123,  154,  234,  251,  257,  307 

Vollery,  28 

Voltaire,  19 

Vorhees,  138,  141,  193,  203,  207,  238, 

246,  247,  318 
Vorhees  and  Lipman,   138,    193,  207, 

238,  246,  247,  318 


W 


WAGNER,  131,  151,  245 

Waksman,  170 

Walton,  259,  264,  282 

Ward,  291,  345 

Warington,  130,  145,  153,  210,  215,  218, 

220,  230,  231,  238,  245 
Warnbold,  276 
Washburn,  387,  389,  390 
Washburn  and  Done,  390 
Wassilieff,  309 
Waynick,  200,  283 
Weichselbaum,  55 
Weinzierl  and  Newton,  393,  394 
Weiss,  412 
Weissenfield,  388 

Welbel,  153,  218,  231,  232,  234,  283 
Welbel  and  Winkler,  232,  283 
Wenner,  56 
Westgate,  315 
Wheeler,  62,  344 

Whipple,  337,  345,  363,  364,  365,  367 
Whipple  and  Mayer,  345 
White,  219 
Whiting,  294,  303,  304,  306,  307,  308, 

312,  318 

Whitson,  232,  234 
Wigand,  292 
Wilbur  and  Ophuls,  401 
Wiley,  218,  410 
Wiley  and  ElweU,  218 
Wilfarth,  27,  248,  291,  292 


424 


INDEX  OF  AUTHORS 


Wilhemy,  197 

Williams,  44,  232,  268,  283 

Wilson,  123,  197,  204,  313,  373 

Windas  and  Knoop,  269 

Winkler,  232,  283 

Winogradsky,  27,  53,  153,  159,  179, 
180,  211,  214,  215,  216,  220,  221,  222, 
225,  250,  252,  264,  272,  307 

Winogradsky  and  Omelianski,  159 

Winslow,  344,  359 

Withers,  139,  158 

Withers  and  Fraps,  139 

Wohl,  84 

Wohltmann,  153 

Wojtkiewiez,  235 


Wollny,  128,  141,  153 
Wood,  415 
Woronin,  291,  292 
Wright,  145 


ZEIT,  103,  104 

Zetnow,  41,  43 

Zinsser,  91,  99 

Zipfel,  293,  295,  299,  313 

Zopf,  55 

Zuber,  190 

Zuco,  210 


SUBJECT  INDEX. 


ABIOGENESIS,  19 

Acid,  acetic,  production  of,  by  bacteria, 

85,  411,  412 
butyric,  production  of,  by  bacteria, 

86 

carbonic,  influence  on  calcium  car- 
bonate, 172 

citric,  production  of,  by  mold,  87 
formic,  produced  by  bacteria,  85,  87 
gluconic,  from  dextrose,  86 
hippuric,  action  of  bacteria  on,  92 
lactic,  mechanism  of  formation,  86 

production  of,  by  bacteria,  85 
oxalic,  produced  by  molds,  87 
phosphates,  action  on  calcium  car- 
bonate, 172 

propionic,  from  propyl  alcohol,  86,  87 
sulphuric,  produced  by  sulphur  bac- 
teria, 179 

uric,  action  of  bacteria  on,  91 
valeric,  produced  by  molds,  87 
Acid-forming  bacteria  in  milk,  376 
Acids,  amino-,  from  proteins,  86 
influence  on  denitrification,  241 

on  potassium  of  soil,  180 
produced  by  Azotobacter,  269 
by  bacteria,  84 

in  soil,  172 

in  cellulose  fermentation,  328,  330 
in  milk,  376 

by  nitrifying  organisms,  176,  178 
Actinomyces  in  soil,  169 
Aeration,  influence  on    ammonia    pro- 
duction, 202,  203 
on  Azotobacter,  281 
on  denitrification,  242,  246 
on  nitrification,  230 
on  number  of  bacteria  in  soil,  162 
on  Ps.  radicicola,  312 
on  toxicity  of  arsenic  in  soil,  124 
Aerobacter,  251 
Aerobes,  69,  109 
Air,  bacteria  in,  336,  339 

inspired  and  expired,  339 
factors  governing  number  and  kind 

of  bacteria  in,  336,  337,  338 
how  bacteria  enter,  336 
Air-borne  infection,  339 
Alcohol  as  disinfectant,  111,  112 


Alcoholic  fermentation,  83,  411 

reactions  of,  83,  84 
Algae,  blue-green,  30,  31 

relationship  to  Azotobacter,  274 
Alinit,  250,  251,  285 
Alkali,  influence  on  nitrification,  220 
Amaneta  muscaria  poisoning,  396 
Amidases,  production  of  ammonia  by, 

206 

Amino-acids,  alcohol  from,  84 
ammonification  of,  206 
in  Azotobacter,  268 
formation  of,  by  Azotobacter,  270 
in  legume  nodules,  309,  310 
Amins,  formation  of,  by  bacteria,  90 
Ammonification,  27,  34,  92,  194-207 
by  actinomyces,  170 
chemistry  of  process,  204-207 
fungi-producing,  1§7 
influence  of  aeration  on,  202,  203 
of  antiseptics  on,  130,  135 
of  arsenic  on,  119 
of  calcium  carbonate  on,  139 

chloride  on,  142 
of  carbohydrates  on,  199 
of  climate  on,  200 
of  crop  on,  200 
of  green  manure  on,  156-159 
of  gypsum  on,  142 
of  iron  sulphate  on,  142 
of  lime  on,  141 

and  magnesia  on,  203 
of  manganese  salts  on,  143,  144 
of  manure  on,  153,  154,  155,  159 
oTmoisture  on,  200,  201 
of  phosphorus  on,  203,  204 
of  potassium  salts  on,  144,  145 
of  season  on,  200 
of  sodium  salts  on,  145 
of  soil  on,  200 
of  water  on,  154,  155 
methods  of  studying,  198 
organisms  concerned  in,  194,  195 
reactions,  192,  193 
stimulation  by  salts,  147 
toxicity  of  salts  on,  149 
variation  with  substrate,  199 
Ammonifiers,  distribution,  196,  197 
Amygdalin,  80 
Amylobacter,  328,  330,  332 
Anabolism  of  Azotobacter,  269 


426 


INDEX 


Anabolism,  bacterial,  7 1 

Ana rrobes,  69 

Animals  and  plants,  differences  in,  29 

Anthrax,  organism  discovered,  24 

vaccine,  25 

Antiseptic  action  on  ammonifiers,  130 
on  bacterial  activities  of  soil,  127, 

128 

on  oxidation  in  soil,  130 
on  plants,  128 
on  soil,  127 

theories  concerning,  133-138 
Grieg  Smith's,  138 
Hiltner's     and     Stormer's, 

133 

Koch's,  133 
Russell's  and  Hutchinson's, 

135,  136 

definition  of,  110 
Antitoxins,  415 
Arsenic,  action  in  soil,  120 
influence  of     aeration  of    soil     on 

toxicity  of,  125 
on  ammonification,  119 
on  azofication,  120 
on  Azotobacter,  261 
on  bacterial  activities,  118-126 
on  nitrification,  119 
on  soil  phosphorus,  122 
occurrence  in  soils,  1 18 
solubility  of,  118 
Ash  of  bacteria,  69 
Aspergillus,  influence  of  salts  on,  67 
Azofication,  248-289 
definition  of,  249 
history  of,  248-252 
influence  of  aeration  on,  281,  282 
of  climate  on,  283-284 
of  colloids  on,  260,  261 
of  crop  on,  282,  283 
of  light  on,  281 
of  manure  on,  266,  267 
of  reaction  on,  254-256 
of  season  on,  281,  282 
of  temperature  on,  278-281 
of  water  on,  276-278 
methods  of  studying,  272-274 
Azofiers,  distribution  of,  252-254 
food  requirements  of,  256-259 
influence  of  organic  substances  on, 

259-261 

metabolism  of,  267-271 
morphology  of,  271,  272 
pigment  production  by,  270,  27 1 
relationship  to  nitrate  accumulation, 

284 

to  other  microorganisms,  274-276 
soil  gain  in  nitrogen  from,  287-289 

inoculation  with,  284-287 
source  of  energy  for,  261-266 
Azotobacter  agilis,  251 

occurrence  in  water,  254 
ash  of,  268 


Azotobacter  beijerinckii,  251 

influence  of  manure  on,  266 
calcium  requirements  of,  259 
carbonic  acid  generated  by,  177 
chroococcum,  251 

calcium  carbonate  on,  255 

formation  of    calcium    carbonate 
by,  173 

humates  on,  265 

manure  on,  266 

methods  of  growing,  272 

morphology  of,  271 

pigments  of,  270,  271 

physiology  of,  272 

radium  rays  on,  103 

soil  inoculation  with,  284-287 
composition  of,  59,  60 
distribution,  252,  253 
food  of,  256-259 
index  of  lime  requirements,  254 

of  phosphorus  requirements,  258 
influence  of  aeration  on,  281 

of  aluminum  on,  259 

of  antiseptic  on,  137 

of  arsenic  on,  121,  122,  261 

of  carbon  bisulphid  on,  128 

of  carbonates  on,  254-257 

of  climate  on,  283 

of  colloids  on,  259 

of  drying  on,  106,  276 

of  green  manures  on,  157 

of  humus  on,  266 

of  light  on,  281 

of  manganese  on,  259 

of  nitrates  on,  256,  257 

of  phosphorus  on,  176,  177 

of  season  on,  281,  282 

of  sodium  on,  256 

of  temperature  on,  278,  279,  280 

of  water,  276,  277 
in  fallow  soil,  335 
iron  requirements  of,  259 
isolation  of,  251 
life  cycle  of,  272 
metabolism  of,  267-270 
nitrogen  of,  268 

fixed  in  different  media  by,   273, 

274 

phosphorus  requirements  of,  257-259 
potassium  requirements  of,  257 
products  of,  86 

relationship    to    cellulose    ferments 
257 

to  higher  plants,  276 

to  other  organisms,  274-276 
soil  gains  in  nitrogen  due  to,  289 
sulphur  requirements  of,  258,  259 
symbiosis  amongst,  276 
utilization  of  cellulose  by,  263 
vinelandii,  251 

composition  of,  60 

forms  of  nitrogen  in,  267,  268 

nitrogen  fixed  by,  264 


SUBJECT  INDEX 


427 


Azotobacter   vinelandii,    pigments   of, 

270,  271 
vitrium,  251 

influence  of  manure  on,  266 
woodstownii,  251 


B 


"BABES  ERNST"  granules,  43 

Bacilli,  37 

Bacillus,    aceto-ethylicum,    formation 

of  acetone  by,  411 
acidophilus,  in  ensilage,  413 
aeris  munitissimus,  in  sewage,  361 
amylobacter,    nitrogen   fixation   by, 

251 
amylozyme,    formation    of    butyric 

acid  by,  411 
anthracis,  action  of  salt  on,  67,  408 

influence  of  pressure  on,   107 

temperature  relations,  97,  98 
aquatilis,  in  water,  346 
asteroporus,  nitrogen  fixer,  251 
aurantiacus,  346 
azophile,  267 

bifermentans  sporogens,  190 
botulinus,  400,  401 

acid  production  by,  86 

influence  of  salt  on,  408 

morphology  of,  400 
bulgaricus,  370,  377 

in  silage,  413 
centropunctatum,  as  denitrifier,  241 

243 
cereus,  167,  168 

cultural  characteristics,  167,  168 

morphology  of,  167 

physiology  of,  168 

in  soil,  165 

cholerae,  action  of  salt  on,  67 
circulans  in  sewage,  361 

in  water,  346 
cloacae  in  canned  beef,  394 

in  sewage,  361 
coagulans,    survive    pasteurization, 

392 
coli,  243 

action  of  copper  on,  123 
on  phosphates,  174 

aerogens  in  cheese,  390 

communis  in  milk,  376,  377 

in  eggs,  393 

indol  and  skatol  production  by,  89 

products  formed  by,  85 

as  putrefier,  190 

in  sausage,  394 

in  water,  346 

weight  of,  64 
cyanogenes  in  milk,  376 

pigment  production  of,  93 
delicatulus  in  sewage,  361 
denitrificans,  240,  241 


Bacillus  denitrificans,  enzymes  of,  244 

longevity  of.  245 
detrudens  in  cheese,  394 
diphtherise  in  butter,  389 

influence  of  salt  on,  408 

in  milk,  377 

temperature  relations,  98 
diplococcus  griseus  in  putrefaction, 

190 
dysenterise,  Flexner,  in  milk,  377 

Shiga,  in  milk,  377 
ellenbachensis,  250 

action  on  cyanamid,  221 
enteritidis  in  food,  397 

in  water,  346 
erythrogenes  in  milk,  376 
ethaceticus,  alcohol  produced  by,  83, 

411 

fecalis  in  sausage,  394 
ferrugineus,  333 
filefaciens,  as  denitrifier,  241 
fitizianus,  alcohol-producing,  83,  411 
flitrovorum,  as  denitrifier,  243 
fluorescens,  action  on  cyanamid,  221 

in  eggs,  393 

liquefaciens,  carbon  requirements, 

242 

denitrifier,   241,   243 
in  water,  346 

non-liquefaciens,  in  water,  346 

pigment  production  by,  93 

in  sewage,  361 
fulvus  in  water,  346 
fuscus,  361 

hartlebi,  denitrifier,  241,  242,  243 
helvolus  in  sewage,  361 
hyalinus  in  sewage,  361 
icteroides,  ammonia-producing,  196 
janthinus,  pigment  production  by,  93 
kirchneri,  action  on  cyanamid,  221 
lactimorbimic  melitensis,  in  milk,  377 
lactis  acidi,  in  milk,  376 

aerogenes  in  water,  346 

viscosus  in  milk,  376 
nitrogen  fixer,  251 
leprse,  nitrogen  requirements,  68 
levaniformus,  nitrogen  fixer,  251 
licheniformis  in  string  beans,  394 
liquefaciens  in  sewage,  361 
liquidus  in  sewage,  361 
megatherium,  165,  166 

action  on  cyanamid,  221 

ammonia  produced  by,  196 

cultural  characteristics  of,  165 

denitrifier,  241 
.  in  foods,  394 

morphology  of,  165 

physiology"  of,  165,  166 

in  soil,  165 
mesentericus,  in  bread,  392 

in  cheese,  394 

in  eggs,  393 

nitrogen  fixer,  251 


428 


SUBJECT  INDEX 


Bacillus  mesentericus,  survive  pasteur- 
ization, 392 
vulgatus,  196 

in  sewage,  361 
methanicus,  334 
monadiformis  in  sewage,  361 
mucosus  in  eggs,  393 
mycoides,  166,  167 
action  on  cyanamid,  221 
on  phosphates,  174 

ammonia  produced  by,  194-198 
cultural  characteristics  of,  167 

denitrifier,  241,  242 

morphology  of,  166 

optimum  conditions  for,  195 

physiology  of,  167 

in  soil,  165 

nibilus  in  sewage,  361 
nitrator,  225 

nitrovorum,  denitrifier,  241 
ochracens  in  water,  346 
orthobutylicus,  formation  of  butyric 

acid  by,  411 
pammellii  in  cheese,  394 
paratyphosus,  in  food,  397 

in  milk,  377 

perfringens  in  putrefaction,  190 
pestis,  influence  of  salt  on,  408 
phosphorescens,  influence  of  temper- 
ature on,  96,  98 
pneumonse,  nitrogen  fixer,  251 
prodigiosus,  denitrifier,  241,  243 

in  milk,  376 

nitrogen  fixer,  251 

pigment  produced  by,  93 

in  water,  346 

proteus,  indol  and  skatol  formation 
by,  89 

in  putrefaction,  190 

vulgaris,  action  on  phosphates,  174 
ammonia-producing,  196 
denitrifier,  241 

in  water,  346 

zenkeri,  as  denitrifier,  241 

in  sewage,  361 

pseudodiphtherise,  influence  of  press- 
ure on,  107 

punctatus  in  water,  346 
putidum,  action  on  cyanamid,  221 
putidus  in  putrefaction,  190 
putrificus  in  putrefaction,  190 
pyocyaneus,  carbon  requirements  of, 
242 

denitrifier,  241 

pigment  produced  by,  93 
radicicola  (See  Ps.  radicicola) 

nitrogen  fixer,  251 
ramosus,  ammonia-producing,  196 

in  eggs,  393 

non-liquefaciens,  as  denitrifier,  241 
rubefaciens  in  water,  346 
ruber  in  water,  346 
rubescens  in  water,  346 


Bacillus  simplex  in  soil,  165 
sporogenes  in  sewage,  361 
stillatus  in  sewage,  361 
stutzeri,  as  denitrifier,  241 
subtilis,  action  on  cyanamid,  221 

on  phosphates,  174 
in  corn,  394 
as  denitrifier,  241,  243 
physiojogical  solution  for,  142 
in  soil,  165 

survives  pasteurization,  392 
temperature  relationship  of,  98 
in  water,  346 
tenus  in  cheese,  394 
tetani,  acid  produced  by,  86 
thermophilis,  temperature  relations 

of,  96,  98 

tumescens,  ammonia-producing,  196 
typhosus,  acid  produced  by,  85 
action  of  copper  on,  123 
in  cheese,  390 
in  cream,  391 
as  denitrifier,  241 
in  ice,  350 
influence  of  freezing  on,  100 

of  salt  on,  408 
longevity  of,  in  butter,  387 
in  milk,  377 
in  sewage,  364 
in  water,  344 
vilatis  in  spinach,  394 
violaceus,  pigment  production  by,  93 

in  water,  346 
viscosus  in  cheese,  394 
vulgaris,  action  on  cyanamid,  221 
ammonia-producing,  196 
in  putrefaction,  190 
vulgatus,  ammonia-producing,  196 

in  corn,  394 

weichselbaumii  in  sewage,  361 
welchii  in  corn,  394 
zopfi  lepsiense,  action  of,  on  cyana- 
mid, 221 

in  putrefaction,  190 
Bacteria,  acid  production  by,  84 
action  of  drying  on,  105 
on  fats,  87 
on  hippuric  acid,  92 
on  minerals,  92 
on  proteins,  87,  88,  89 
on  sulphur,  175,  178 
on  urea,  91 
on  uric  acid,  91 
in  air,  336-339 
expired,  339 
inspired,  339 
methods  of  entering,  336 
number  and  kind  of,  336,  337 
amins,  formation  of,  90 
in  arts  and  industries,  411,  416 
autotrophic,  67 
in  body,  31  32 
in  bread,  392 


SUBJECT  INDEX 


429 


Bacteria,  Brownian  movement  of.  40, 

41 

in  butter,  387 
in  canned  foods,  394 
capsules  of,  43 
carbohydrates  in,  58 
carbon  requirements  of,  68 
cellulose  in,  42 
changes  produced  in  milk  by,  375, 

376 

in  cheese,  289,  390 
chitin  in,  43 
classes  of,  in  milk,  376 

in  water,  345,  346 
classification  of,  46-57 
composition  of,  58-62 
cytoplasm  of,  43 
definition  of,  29 
discovery  of,  17 
in  eggs,  393 
in  ensilage,  413 
energy  for,  65 
extractives  in,  58 
factors  influencing  number  and  kind 

in  soil,  162,  164,  337,  338 
food  requirements  of,  63-70 
gradation  of,  38 
hemicellulose  in,  42 
hydrolyzing  in  sewage,  362 
hydrogen  requirements  of,  68 
in  ice  cream,  390,  391 
indol,  production  by,  89 
influence  of  antiseptics  on,  129-138 

of  calcium  carbonate  on,  139 
chloride  on,  142 

of  chemicals  on,  108-117 

of  cold  on,  100 

of  electricity  on,  103 

of  gypsum  on,  142 

of  heat  on,  95 

of  iron  sulphate  on,  142 

of  light  on,  95,  101,  105 

of  lime  on,  141 

of  magnesium  salt  on,  143 

of  manganese  salts  on,  143,  144 

of  manure  in  soil,  151,  153,  154 

of  moist  heat  on,  99 

of  moisture  in  soil  on,  154,  155 

of  osmotic  pressure  on,  106 

of  oxygen  on,  105 

of  potassium  salts  in  soil  on,  144, 
145 

of  radium  rays  on,  103 

of  Rontgen  rays  on,  103 

of  salts  on,  139 

of  shaking  on,  107 

of  sodium  salts  in  soil  on,  145 

of  temperature  and  light  on,  9o-102 
involution  forms,  38 
as  liberators  of  phosphorus,  174 
longevity  of,  45 
in  lungs,  32 
in  meat,  393 


Bacteria,  metachromatic  granules,  43 
method  of  determining  number  of, 

160 
in  milk,  31,  372,  373 

certified,  371 

common,  372 

condensed,  391,  392 

sources  of,  372,  373 

speed  of  growth  in,  374,  375 
moisture  in,  65 

content  of,.  58 
morphology  of,  37-45 
motility  of,  17,  42 
nitrogen  requirements  of,  69 
occurrence  of,  31 
oxygen  requirements  of,  69 
phosphorus  requirements  of,  69 
pigment  production  of,  93 
in  plants,  32 
pleomorphism  of,  38 
potassium  requirements  of,  69 
production  of  heat  by,  94 

light  by,  94 

ptomains  produced  by,  91 
r61e  in  nature,  32 
in  sewage,  361 

oxidizing  in,  363 

pathogens  in,  364,  365,  366 

reducing  in,  363 
sheath  of,  43 
skatol  formation  by,  89 
soil  formers,  33,  171,  172 
in  soils,  kinds  ©f,  164,  165 

number  of,  161,  162 
spores  of,  44 
stimulation  by  salts,  147 
in  stomach,  32 
sulphur  requirements  of,  69 
toxicity  of  salts  for,  149 
vital  movement  of,  41 
vitamine  requirements  of,  70 
in  water,  342 

classes  of,  345,  346 

influence  of  light  on,  343 

of  sedimentation  on,  343 

of  temperature  on,  344 
weight  of,  39 
zoogloea,  43 
Bacterial    activities,     influence    of 

arsenic  on,  118-126 
of  enzymes  on,  71-81 
of  manure  on,  150-159 
of  salts  on,  139-149 
counts,  value  of,  161 
metabolism,  71-94 

products  of,  81-94 
toxins,  91 

Bacteriology,  agricultural,  27,  36 
dairy,  36 
definition  of,  29 
development  of,  17-28 
industrial,  36 
pathological,  36 


430 


SUBJECT  INDEX 


Bacterium,  acetic  acid  produced  by 

85 

chrysogloea,  nitrogen  fixer,  252 
diphtheria?  in  milk,  377 
lactis  aerogenes  in  milk,  376,  377 
lactus  acidi  in  sauerkraut,  412 
lipsiense,  nitrogen  fixer,  252 
pasteurianum,  acid  produced,  85,  86 

morphology  of,  411 
pneumonia?,  alcohol-forming,  83 
schutzenbachi,  morphology  of,  412 
tartaricus,  nitrogen  fixed  by,  252 
termo,  in  putrefaction,  190 
tuberculosis,  in  butter,  388 

composition  of,  59 

influence  of  salt  on,  408 

in  milk,  377,  382 

in  oleomargarine,  389 

in  sewage,  364 

temperature  relation  of,  96,  98 
Bacteroids,  291,  302,  303 


CALCIUM  carbonate  formed  in  soil,  173 
losses  in  soil,  172,  173 
on  soil  flora,  139 
transformation  in  soil,  172,  173 
Capsules,  43 

composition  of,  61 

Carbohydrates,  action  of,  in  soil,  246 
in  bacteria,  58 
influence  on  ammonia  production  in 

soil,  199 

on  denitrification  in  soil,  242,  243 
on  nitrification  in  soil,  222 
on  nitrogen  fixation  in  soil,  262 
products    formed   from,    by  Azoto- 

bacter,  268 

as  source  of  carbon,  68 
Carbon  bisulphid,  action  of,  on  soil,  127, 

128 

on  Azotobacter,  128 
on  plants,  127 

compounds,  influence  of,  on  denitrifi- 
cation, 243 
cycle,  182,  183 

nitrogen  ratio  influence  on  nitrogen 
fixation  by  Azotobacter,  263 
264 

in  soil,  191 

sources  of,  for  bacteria,  68 
for  nitrifiers,  220,  221 
for  plants,  290 
Catalyzers,  definition  of,  75 

as  ferments,  71,  73 
Canned  food,  bacteria  in,  394 
Canning,  407 
Cellulose,  327 
in  bacteria,  42 

decomposing  ferments,  327-335 
early  observations  on,  327-330 


Cellulose  decomposition  by  actinomy- 

cetes,  170 

ferments,  aerobic,  333 
function,  333,  334,  335 
involution  forms  of,  333 
isolation  of,  330,  331 
morphology  of,  331-333 
products  formed  by,  332 
Decent  work  on,  333 
in  sewage,  363 

temperature  relations  of,  333 
sources  of  energy  for  Azotobacter, 

263 

Cell  wall  of  bacteria,  42 
Cheese,  bacteria  in,  389,  390 
Chemical  preservation   of  foods,   408, 

409 
Chemicals,  influence   of,  on    bacteria, 

108-117 

Chemotaxis,  108,  109,  304 
Chitin  in  bacteria,  43 
Chlorinated  lime  as  disinfectant,  1 15 
Chlorine  compounds  as  disinfectants, 

114 
Cholera  due  to  water,  351,  352,  354 

vibrio,  composition  of,  61 
Classification  of  bacteria,  46-57 
bacterial  products,  82 
difficulties  of,  46,  48 
from  food  requirements,  67 
Jensen's,  67 
Migula's,  46 
in  regard  to  heat,  96 
Soc.  Am.  Bact.,  50 
of  bacterial  enzymes,  79 

pigments,  93 

Linnaean  system  of,  for  plants,  48 
of  waters,  340,  341 

Climate,  influence  of,  on  nitrogen  fixa- 
tion, 283,  284 
Clostridium,  86 

americanum,  nitrogen  fixed  by,.  264 

utilization  of  cellulose  by,  263 
gelatinosum,  241 

nitrogen  liberated  by,  243 
pasteurianum,  discovery  of,  250 
method  of  growing,  272 
morphology  of,  252,  271 
nitrogen  fixed  by,  264 
occurrence  of,  in  water,  254 
physiology  of,  272 
symbiosis  with  Azotobacter,  276 
Cocci,  37 
Cold,  influence  of,  on  pathogens,  406 

preservation  of  food  by,  405 
Colloids,  influence  on  Azotobacter,  260, 

261 
Composition  of  bacteria,  58-61 

of  water  bacillus,  60 
Copper,  action  of,  on  soil  bacteria,  122, 

123 

in  food,  397 
Crenothrix  polyspora,  180 


SUBJECT  INDEX 


431 


Crop,   influence  of,  on  bacterial  flora, 

250 

on  nitrates  of  soil,  236 
on  nitrification,  230,  231 
on  nitrogen  fixation,  282,  283 
production,  essential  elements  in,  319 
rotation,  319-326 
Cycle,  biological,  in  sewage,  363 
of  carbon,  182,  183 
of  elements,  181-187 
of  nitrogen,  183,  184 
of  phosphorus,  184,  187 
of  sulphur,  184 
Cytoplasm,  composition  of,  61 


DECAY,  188-193 
definition  of,  188 
products  of,  190,  191 
Denitrification,  239-247 
by  actinomyces,  170 
early  theories  on,  239,  240 
enzymes  concerned  with,  81 
influence  of  media  reaction  on,  241, 

242 

of  temperature  on,  245 
.     of  water  on,  244,  245 
losses  of  nitrogen  in,  245,  246,  247 
organisms  concerned  in,  240,  241 
reactions  of,  92 
Denitrifiers,  food  requirements  of,  242, 

243 

function  of,  247 
metabolism  of,  243,  244 
Deodorants,  110 
Desiccation,  influence  on  Azotobacter, 

276 

Diplococci,  37      x 
Disease  due  to  meat,  397 
to  milk,  378 
to  water,  351,  352 
milk-borne,  character  of,  379,  380 

extent  of,  380-383 

Disinfectant,  chlorinated  lime  as,  1 15 
definition  of,  110 
formaldehyd  as,  116 
hydrocyanic  acid  as,  1 17 
mercuric  chlorid  as,  117 
sulphur  dioxid  as,  116 
Disinfectants,  chlorin  compounds  as, 

114 

classes  of,  111 
emulsions  as,  113,  114 
influence  of  medium  on,  113 
of  moisture  on,  111 
of  temperature  on,  111 
laws  governing  action  of,  111 
mode  of  action,  111 
Drying,  preservation  of  food  by,  406 
Dysentery  due  to  water,  353 


E 

ECTOPLASM,  42 

composition  of,  61 
Eggs,  bacteria  in,  392,  393 
Electricity,  germicidal  influence  of,  on 

bacteria,  104 
influence  of,  on  bacteria,  103 

on  medium,  104 
Emulsion,  80 

Energy,  liberation  by  enzymes,  80 
sources  of,  64 

for  Azotobacter,  261-266 
for  denitrifiers,  243 
for  nitrifiers,  223 
Ps.  radicicola,  312 
Ensilage,  413,  414 
Enzymes,  71-81 

action  of,  on  phosphates,  175 

classification  of,  79 

concerned  in   ammonification,    204, 

206 

in  cellulose  fermentation,  330 
in  denitrification,  243 
in  nitrification,  224 
in  Ps.  radicicola  metabolism,  305 
definition  of,  72,  73 
factors  governing  action  of,  76-79 
hydrolytic,  79 
influence  of  poisons  on,  79 
of  temperature  on,  77,  79 
of  time  on,  78 
oxidizing,  81 
properties  of,  76 
reversible  action  of,  78 
specificity  of,  77 
terminology  of,  75,  76 
Epidemiologist's  method  of  working, 

355-358 
Extractives  in  bacteria,  58,  59 


F 


FALLOW,  influence  of,  on  nitrification, 
231-235 

loss  of  nitrates  from,  231 
Fats,  action  of  bacteria  on,  87 
Fermentation,  188-193 

of  alcohol,  83,  84 

alcoholic,  411 

definition  of,  188 

early  theories  of,  21,  71,  72 

enzymes  of,  79 
Ferments,  extracellular,  72 

intracellular,  72 

organized,  72 

unorganized,  72 
Fertilizers,   influence  of,   on  legumes, 

313,  314 
Food,  bacteria  in-,  387-394 

function  of,  for  bacteria,  64 

milk  as,  368-370 

preservation  of,  404-410 


432 


SUBJECT  INDEX 


Food,  preservation  of,  by  canning,  407 
by  chemicals,  408,  409 
by  cold,  405 
by  drying,  406 
importance  of,  404 
methods  of,  404,  405 
pressure,  406,  407 
by  sugar  and  salt,  407,  408 
pure,  law,  409,  410 
requirements  of  bacteria,  63-70 
maximum,  64 
minimum,  63 
Food-poisoning,  395-403 
botulism,  400,  401 
classes  of,  305 

diseased  animals  causing,  397 
foods  causing,  398,  399 
metallic,  396,  397 
paratyphoid  causing,  397,  398 
prevention  of,  402,  403 
ptomain,  400 

Formaldehyd  as  disinfectant,  116 
Freezing,  influence  of,  on  bacteria,  100 

in  soil,  162,  163 

Fruits,  preservation  of,  by  pressure,  107 
Fungi,  ammonia  production  by,  197 

filamentous,  nitrogen  fixers,  252 
Fusel  oil,   produced  in  alcoholic  fer- 
mentation, 84 
Future  work  in  bacteriology,  27 


GASES,  in  cellulose  fermentation,  329, 

332,  333,  334 

Germicidal  action  of  moist  heat,  99 
Germicide,  110 
Glucose,  alcohol  from,  83,  84 

energy  from,  65 

lactic  acid  from,  86 

succinic  acid  from,  86 
Glycogen  in  bacteria,  62 
Gonococcus,  37 
Gradation  of  bacteria,  38 
Granules,  "Babes-Ernst,"  43 

metachromatic,  43 
Granulobacter,  251 


HEAT,  influence  of  moist,  on  bacteria, 

98,  99 

on  nitrifiers,  226 
production  of,  by  bacteria,  94 
relationship  of  bacteria  to,  96 
Hemicellulose  in  bacteria,  42 
Humates,  influence  of,  on  nitrogen  fixa- 
tion, 265. 
Humus,  chemistry  of  formation  pf;  192, 

1  .  *• ) 

formation  of,  190 


Humus,  formation  of,  by  cellulose  fer- 
ments, 333 

influence  of,  on  Azotobacter,  266 
on  nitrification,  227 
on  water  requirements  in  nitrifi- 
cation, 227 

value  of,  in  soil,  191,  192 
Hydrocyanic  acid,  117 
Hydrogen  requirements  of  bacteria,  68 
Hydrolytic  enzymes,  79 


ICE,  bacteria  in,  350 

cream,  bacteria  in,  390 

disease  due  to,  3£  9 
Indican,  formation  of,  90 
Indol,  formation  of,  89 
Infection,  air-borne,  339 

caused  by  food,  399 

sources  of,  in  milk,  378,  379 
Inorganic  constituents  in  Bacteria,  61 
Insecticide,  hydrocyanic  acid  as,  117 

sulphur  dioxid  as,  116 
Involution  forms  of  bacteria,  38 
Iron,  action  of  bacteria  on,  92 

bacteria,  180 

influence  of,  on  nitrification,  219 

requirements  of  Azotobacter,  259 


KATABOLISM,  bacterial,  71 
Keffir,  411 
Kinase,  76 
Kumiss,  411 


LEAD,  influence  of,  on  soil  bacteria,  122 
Leben,  411 

Lecithin,  hydrolysis  of,  174 
Legumebacter,  species  of,  295;  296,  297 
Legumes,    chemical    composition    of, 
308-312 

elements  added  to  soil  by,  319,  320 

feed  on  nitrates,  323,  324 

influence  of,  on  non-legumes,  314,  315 

immunity  to  Ps.  radicicola,  305 

methods  of  assimilating  nitrogen  by, 
307 

nitrogen  in,  323 

power  to  fix  nitrogen,  291 

sources  of  nitrogen  for,  322,  323 
Life  cycle  of  Azotobacter,  272 
Light,  influence  of,  on  Azotobacter,  281 
on  bacteria,  101,  105 
on  denitrifiers,  245 

production  of,  by  bacteria,  94 
Lime,  influence  of,  on  ammonificatioi 

203 


\ 


SUBJECT  INDEX 


433 


Lime,  influence  of,  on  nitrification,  219 

on  nitrogen  fixation,  255 
requirements    of    soil,    Azotobacter 

as  an  indicator  of,  254 
Lipases,  80 
Listerism,  26 
Longevity  of  bacteria,  45 


M 


MAGNESIUM  carbonate,  influence  of,  on 

nitrification,  219 

salts,  influence  of,  on  bacteria,  143 
Maltose,  79,  80      , 

Manganese  salts,  influence  of,  on  bac- 
teria, 143,  144   ( 

Manure,  denitrifying  organisms  in,  240 
green,  ill  effects  from  use  of,  158 

influence  of,  on  soil,  152,  156,  159 
influence  of,  on  ammonification,  153 
on  Azotobacter,  266,  267 
on  bacteria,  151-153 
on  bacterial  activities,  150-159 
on  moisture  of  soil,  151 
on  nitrification,  153 
on  nitrogen  fixation,  153,  154 
on  temperature  of  soil,  151 
products  of  decomposition,  330 
Mass  and  enzyme  action,  78 
Matzoon,  411 

Micrococcus  acidi  in  cheese,  394 
albicans  amplus,  361 
candicans  in  beef,  394 
candidus  in  baked  beans,  394 
casei  in  sewage,  361 
cereus  in  beans,  394 
fervidosus  amplus  in  sewage,  361 
flavus  liquefaciens  in  putrefaction, 

190 
gonorrhese  nitrogen  requirements,  68 

temperature  relationship  of,  98 
lactis  in  cheese,  394 
luteus  in  corn,  394 
meningitidis,  vitamine  requirements 

of,  70 

pyogenes  in  corn,  394 
stellatus  in  beef,  394 
tetragenus  mobilis  ventriculi,  361 
ureae,  81 
Micron,  39 

Microorganisms,  kinds  of,  in  soil,  164 
Microspira  sestuarii,  action  of,  on  sul- 
phates, 179 

desulphuricans,    action   of,    on  sul- 
phates, 179 

Mills  Reincke  phenomenon,  353 
Milk,  abnormal  changes  in,  376 
acid-forming  bacteria  in,  376,  377 
bacteria  in,  372 
bacteriology  of,  368-377 
bitter,  376 
certified,  371 
28 


Milk,  changes  produced  in,  by  bacteria, 
375-376 

classes  of,  371 
of  bacteria  in,  376,  377 

common,  371 

composition  of,  368 

disease  and,  378-386 

factors  influencing  number  of  bac- 
teria in,  372,  373 

as  food,  368,  369,  370 

growth  of  bacteria  in,  374,  375 

influence  of,  on  intestinal  microflora, 
371 

pasteurization  of,  385,  386 

pathogenic  bacteria  in,  377,  378 

peptonizing  bacteria  in,  ^77 

quantity  consumed,  368 

sources  of  infection  in,  378,  379 

tubercle  bacilli  in,  382 

tuberculosis  due  to,  381 
Milk-borne  disease,  character  of,  379. 

380 

extent  of,  380,  381 
Mineralization    of    soil    constituents, 

171-180 
Moisture  in  bacteria,  58,  65 

function  of,  in  organism,  66 

influence  of  manure  on  soil,  151 

requirements  of  nitrifiers,  226-230 
Molds,  acid  produced  by,  87 

as  denitrifiers,  241 

difference  of,  from  bacteria,  30 

in  eggs,  393 
Morphology  of  Azotobacter,  271 

of  bacteria,  37-45 

of  cellulose  ferments,  331 

of  clostridium  pasteurianum,  271 

of  mtrifiers,  225,  226 
Motility,  organs  of,  42 
Mustard,  as  green  manure,  157 


N 


NITRATE   accumulations,    relationship 

of,  to  Azotobacter,  284 
influence  on  loss  from  soil,  247 
losses  of,  from  soil,  236,  237 

prevention  of,  238 
quantities  formed  in  soil,  153,  158, 

235  236 

used  by  legumes,  323,  324 
Nitrification,  28,  34,  208,  238 
of  calcium  cyanamid,  221 
chemistry  of  process,  223,  224 
discovery   of   organisms   concerned, 

209,  210 
early  knowledge  of,  208 

theories  of,  208,  209 
energy  transformations  in,  223 
influence  of  aeration,  230,  231 

of  antiseptics  on,  134 

of  arsenic  on,  119 


434 


SUBJECT  INDEX 


Nitrification,  influence  of  calcium  on, 

142 

carbonate  on,  139 
of  chloroform  on,  210 
of  crop  on,  231,232,  233,  324,  325 
of  cultivation  on,  230,  231 
of  fallow  on,  231-233 
of  green  manure  on,  156-159 
of  gypsum  on,  142 
of  iron  sulphate  on,  142 
of  light  on,  230 
of  lime  on,  141 
of  magnesium  salts  on,  143 
of  manganese  salts  on,  143,  144 
of  manure  on,  153,  154,  155 
of  moisture  on,  154,  155,  226-230 
of  organic  matter  on,  211,  222 
of  potassium  salts  on,  144,  145 
of  reaction  on,  218,  219 
of  season  on,  234 
of  sodium  salts  on,  144,  145 
of  temperature  on,  230 
of  water-holding  capacity  of  soil 

on,  228,  229 
isolation    of    organisms    concerned, 

211-215 

stimulation  of,  by  salts,  147 
toxicity  of  salts  on,  149 
Nitrifying   ferments,    distribution    of, 

217,  218 

function  of,  in  soil,  215 
influence  of  depth  of  soil  on,  218 

of  heat  on,  226 
isolation  of,  211-215 
metabolism  of,  223,  224 
Nitrite  nitrogen  in  soil,  228 
Nitrobacter,  214,  216 
morphology  of,  226 
sources  of  energy  for,  214,  215 
Nitrogen  in  crops,  320,  321 
cycle,  183,  184 

fixation,  chemical  theory  of,  249,  250 
early  theories  of,  290,  291 
energy  for,  312 
influence  of  aeration  on,  312 
of  arsenic  on,  '120 
of  combined  nitrogen  on,  250 
of  cultivation  on,  283 
of  fertilizers  on,  313 
of  green  manures  on,  156-159 
of  heated  soil  on,  131 
of  manure  on,  151 
of  moisture  on,  312 
of  season  on,  282 
of  temperature  on,  279,  280,  313 
mechanism  of,  306-312 
in  soils,  34,  35 

fixed  by  Azotobacter,  264,  265 
in  legumes,  323,  324 
liberation  of,  by  bacteria,  243 
losses  in  nitrification,  225 
in  nodules,  309 
soil  gains  in,  287,  315,  316,  352 


Nitrogen,  soil  losses  of,  239 

sources  of,  for  bacteria,  68 

in  Utah  soils,  320,  325 
'Nitrogen,"  318 

STitrosococcus,  morphology  of,  226 
STitrosomonas,  influence  of,   on   phos- 
phates, 175 

media  for,  220 

morphology  of,  214,  225,  226 

sources  of  nitrogen  for,  221 
Nodule,  ash  of,  311 

bacterial  growth  of,  304,  305 

composition  of,  309 
Nuclein,  action  of  bacteria  on,  174 


OIDIUM  lactis,  action  of,  on  lactic  acid, 

87 

influence  of  pressure  on,  107 
in  milk,  376 
Oleomargarine,  tubercle  bacilli  in,  389 
Organic   compounds,     action    of,     on 

Azotobacter,  259,  260 
on  denitrification,  243 
in  bacteria,  58 

as  energy  for  Azotobacter,  262,  263 
bacteria,  159 
of  soil,  193 
manures,    speed    of    decomposition, 

199 

Osmotic  pressure,  67 
on  bacteria,  106 
influence  of,  in  soil,  149 
Oxidation,  of  acids,  87 

incomplete,  82 
Oxidizing  enzymes,  79,  81 
Oxygen,  influence  of,  on  bacteria,  105 
requirements  of  bacteria,  69 


PATHOGENIC  bacteria  in  cheese,  390 
Peat,  bacterized,  286 

denitrification  in,  241 
Penicillium,  influence  of  salt  on,  67 
Peroxidases,  81 
Phosphate,  action  of  bacteria  on,  176 

reaction  in  soil,  175 

required  by  zymase,  83 
Phosphoproteins,    action    of    bacteria 

on,  174 

Phosphorus,    action   of    cellulose    fer- 
ments on,  334 

cycle,  184-187 

requirements  of  Azotobacter,   257- 

258 
of  bacteria,  64 

in  soil,  173 

in  Utah  soil,  320 


SUBJECT  INDEX 


435 


Pigment,    production    of,    by    actino- 

mycetes,  169,  170 
by  Azotobacter,  94,  270,  271 
Pigments,  classes  of,  93 

production  of,  93 
Plants,  classes  of,  30,  31 
elements  in,  181 
poisonous,  395,  396 
Plasmolysis,  67 
Pleomorphism,  38 
Pneumococcus,  37 
Poison,  metallic,  396,  397 
Poisoning,  395-403 
classes  of,  395 
food  causing,  398 
from  diseased  meat,  397,  398 
from  ensilage,  414 
prevention  of,  402,  403 
ptomain,  400 
symptoms,  397,  398 
Poisonous  foods,  395,  396 
Polar  bodies,  43 
Potassium  carbonate,  influence  of,  on 

nitrification,  218,  219 
liberation  of,  by  bacteria,  180 
requirements  of  Azotobacter,  257 

of  bacteria,  69 
salts,  influence  of,  on  bacteria,  144, 

145 

in  Utah  soils,  320 
Preservative,  definition  of,  110 
Pressure  on  bacteria,  106,  107 
for  preserving  food,  107 
preservation  of  food  by,  406,  407 
Proteases,  80 

Protein,  action  of  bacteria  on,  88 
hydrolysis,  204,  205 
liquefaction  in  sewage,  362 
as  source  of  carbon,  68 
Protista,  29 

Protozoa,  action  of  antiseptic  on,  131 
of  arsenic  on,  121 
of  heat  on,  131 
Protozoan  theory,  135 
Pseudomonas  fluorescens,  168 
ammonia-producing,  196 
morphology  of,  168 
physiology  of,  168 
in  soils,  165 

nebulosa  in  sewage,  361 
ochracea  in  sewage,  361 
radicicola,  292 
bacteroids  of,  303,  304 
commercial  cultures  of,  318 
cultural  characteristics  of,  297-299 
entrance  into  host,  304 
influence  of  acids  on,  297 
of  aeration  on,  312 
of  drying  on,  299 
of  fertilizers,  313,  314 
of  moisture  on,  312 
of  phosphorus  in  soil,  178 


Pseudomonas   radicicola,   influence   of 

temperature  on,  299,  313 
metabolism  of,  306-308 
morphology  of,  299-303 
relationship  to  host,  305,  306 
sources  of  energy  for,  312 
species,  292-299 
staining  of,  303 
turcosa  in  sewage,  361 
Psychrophelic  bacteria,  96,  97 
Pteridophytes,  30 
Ptomain  poisoning,  400 
Ptomains,  90,  91,  189 
Putrefaction,  188-193 
definition  of,  188 
organisms  concerned  with,  190 
products  of,  190,  191 


RABIES,  26 

Radiobacter,  251 

Radium  rays,  action  of,  on  bacteria,  103 

Rain,  influences  of,  on  soil  nitrates,  236 

Rays,  ultraviolet,  as  catalyzers,  225 

Reducing  enzymes,  79,  81 

Reductase,  81 

Retting  of  flax,  414 

Rhizobium  beijerinckii,  297 
radicicola,  297 

Rontgen  rays,    influence  of,   on  bac- 
teria, 103 

Rotation  and  soil  fertility,  325 

Rothamsted,  321,  322 


S 


i  SALT,  influence  of,  on  bacteria,  67 
I  Salts,  influence  of,  on  bacteria,  139,  147 
on  bacterial  activities  of  soil,  139- 

149 

toxicity  of,  145,  146,  149 
Sarcina  alba  in  sewage,  361 
aurantiaca,  pigments  of,  93 
lutea,  pigment  of,  93 

production  of  ammonia  by,  196 
in  water,  346 
Sauerkraut,  412 
Schizases,  79 
Schutz-Borissow  law,  78 
Season,  influence  of,  on  Azotobacter, 

281,  282 

Sewage,  bacteria  in,  361,  362 
composition  of,  360 
disposal  of,  366,  367 
hydrolyzing  bacteria  in,  362,  363 
oxidizing  bacteria  in,  363 
pathogenic  bacteria  in,  364,  365,  366 
reducing  bacteria  in,  363,  364 
Shaking,  influence  of,  on  bacteria,  107 
Sheath,  43 


436 


SUBJECT  INDEX 


Silage,  temperature  of,  94 

Silkworm  disease,  23 

Skatol,  formation  of,  by  bacteria,  89 

Smallpox,  24 

Sodium  salts,  influence  of,  on  bacteria, 

145 

Soil  actinoinycetes,  169    ( 
arsenic  in,  118 

bacteria,  action  of,  on  proteins,  88 
as  formers,  32,  33,  171,  172 
number  of,  161,  162,  163,  164 
in  water,  346,  347 
biological  changes  produced  in,  by 

bacteria,  150 
definition  of,  171 
flora,  160,  170 
gain  in  nitrogen,  248,  287,  289,  315, 

316 

influence  of  antiseptics  on,  127 
of  carbon  bisulphid  on,  128,  129. 

131 

of  freezing  on,  100,  101 
of  green  manure  on,  156-159 
of  heat  on,  127,  131,  132 
of  manure  on,  150 
inoculation,  284-287 

methods  of,  316-317 
loss  of  nitrates  from,  236,  238 
organic  constituents  of,  193 
phosphorus,  liberation  of,  173-175 
plant  food  in  Utah,  320 
potassium,  liberation  of,  180 
psychrophilic  bacteria  in,  96,  97 
temperature,  151,  154,  280,  313 
Spirillum  chplerse  asiaticse,  97,  98 

desulphuricans,  178 
Spirophyllum  ferrigineum,  180 
Spontaneous  generation,  18,  19 
Spores,  44,  45 
germination,  45 
resistance,  44 
Staphylococci  in  eggs,  393 
Staphylococcus  aureus,  pigment  of,  93 

survives  pasteurization,  392 
pyogenes  albus  in  putrefaction,  190 
Streptococcus    coligracilis   in   sewage, 

361 

enteritis  in  sewage,  361 
lacticus,  absence  of  katalase  in,  81 
acid  produced  by,  85 
in  sauerkraut,  412 
pyogenes  in  putrefaction,  190 
Substrata,  75 

Sugar  and  salt,  as  preservatives,  407 
Sulphates,  action  of  bacteria  on,  93 

reduction  of,  by  enzymes,  81 
Sulphur,   action   of  bacteria  on,   175, 

178 

bacteria,  179 
cycle,  184 

dioxid  as  a  disinfectant,  116 
requirement  of  Azotobacter,  259 
of  bacteria,  69 


Sulphuric  acid,  catalytic  production  of, 

76 
Symbiosis,  306 

among  Azotobacter,  276 


TANNING,  414,  415 

Temperature,  and  coagulation  of  bac- 
terial protoplasm,  96 
fatal,  98 

influence  of,  on  bacteria,  95,  100,  101 
on  enzymes,  79 
on  denitrification,  245 
on  legume  bacteria,  313 
on  manure  in  soil,  151,  154 
on  nitrification,  230 
on  nitrogen  fixed,  279,  280 
on  reactions,  95 
on  soil,  154 
on  water  bacteria,  344 
relationships  of  bacteria,  97 
ThaUophytes,  30,  31 
Thermal  death  point,  99 
Thermophilic  bacteria,  96,  97 

organisms  as  nitrogen    fixers,   279, 

280 

Thiothrix,  179 

Toluene,  action  of,  on  soil,  136 
Toxic  compounds  in  ensilage,  414 
Toxicity  of  salt  for  bacteria,  149 
Toxins,  91 

bacterial,  in  soil,  138,  236 
formation  of,  in  soil,  235 
influence  of  heat  on,  132 
Tryptophan,  action  of  bacteria  on,  89 
Tubercles,  root,  early  observations  on, 

291,  292 

Tuberculin  test,  383,  384,  385 
Tuberculosis  among  cattle,  381,  382 

due  to  milk,  380,  381 
Typhoid  bacilli,  resistance  of,  to  lactic 

acid,  387 
carriers,  399 

cause  of,  how  determined,  355-359 
character  of  outbreak  of,  355 
due  to  milk,  379,  380 

to  water,  351,  352,  354 
para,  outbreaks,  397,  398 


UREA,  action  of  bacteria  on,  91 
Urease,  80 

Uric  acid,  products  from,  91 
Uricarse,  92 


VACCINE,  for  anthrax,  25 
Vaccines,  415 


SUBJECT  INDEX 


437 


Vinegar,  bacteriology  of,  411/412 

oxidase,  81 
Vital  movement,  41 
Vitamin  requirement  of  bacteria,  70 
Vitamins  in  milk,  370 


W 


WATER,  catalytic  action  of,  66 
chemical  purification  of,  349 
cholera  due  to,  352,  353 
classes  of  bacteria  in,  345,  346 
classification  of,  340,  341 
disease  and,  351-359 
diseases  transmitted  by,  352 
drainage,  nitrogen  in,  237 
dysentery  due  to,  353 
ground,  341 
importance' of ,  340 
influence  of,  on  ammonification,  200, 

201 

on  Azotobacter,  276,  277 
on  denitrification,  244 
of  food  on  number  of  bacteria  in, 

344,  345 
of  light  on  number  of  bacteria  in, 

343  344 

on  nitrification,  226-230 
on  nitrogen  fixation,  276-278 
on  Ps.  radicicola,  312,  313 
of  sedimentation  on  bacteria  in, 
343 


Water,    influence  of    temperature   on 

number  of  bacteria  in,  344 
intestinal  bacteria  in,  347 
natural  purification  of,  347,  348 
purification  of,  by  alum,  349 

by  chlorazine,  349 

by  potassium  permanganate,  349 
soil  bacteria  in,  346,  347 
solvent,  66 
stored,  341 
surface,  341 

typhoid  due  to,  354,  355 
Water-holding    capacity    of    soil,    re- 
lationship   of,    to    ammonification, 

201 


YEAST,  action  of,  on  phosphates,  175 

as  denitrifier,  240 

difference  of,  from  bacteria,  30 

nitrogen  fixed  by,  252 

production  of  ammonia  by,  194 
Yellow  fever,  26 


Z 


ZINC,  action  of,  on  bacteria,  123,  124 

Zoogloaa,  43 

Zymase,  phosphorus  requirements     of, 

83 

Zymases,  80 
Zymo-excitor,  76 
Zymogen,  76 


7  BIOLOGY 
UBRARf 
G 


UNIVERSITY  OF  CALIFORNIA  UBRARY 


